US20090133156A1

US20090133156A1 - Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics and phenotypes

Info

Publication number: US20090133156A1
Application number: US12/139,269
Authority: US
Inventors: Nickolai Alexandrov; Vyacheslav Brover; Peter Mascia; Ken Feldmann
Original assignee: Ceres Inc
Current assignee: Ceres Inc
Priority date: 2004-06-30
Filing date: 2008-06-13
Publication date: 2009-05-21
Also published as: US20130139278A1; US7396979B2; WO2006004955A2; WO2006004955A3; US20060057724A1

Abstract

Isolated polynucleotides and polypeptides encoded thereby are described, together with the use of those products for making transgenic plants.

Description

CROSS REFERENCE TO RELATED APPLICATION

This is a Divisional Application of U.S. Nonprovisional application Ser. No. 11/272,740 filed on Jun. 30, 2005 which claims priority under 35 U.S.C. § 119(e) on U.S. Provisional Application No(s). 60/583,621; 60/584,829 and 60/584,800 filed on Jun. 30, 2004, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to isolated polynucleotides, polypeptides encoded thereby, and the use of those products for making transgenic plants or organisms, such as transgenic plants.

BACKGROUND OF THE INVENTION

There are more than 300,000 species of plants. They show a wide diversity of forms, ranging from delicate liverworts, adapted for life in a damp habitat, to cacti, capable of surviving in the desert. The plant kingdom includes herbaceous plants, such as corn, whose life cycle is measured in months, to the giant redwood tree, which can live for thousands of years. This diversity reflects the adaptations of plants to survive in a wide range of habitats. This is seen most clearly in the flowering plants (phylum Angiospermophyta), which are the most numerous, with over 250,000 species. They are also the most widespread, being found from the tropics to the arctic.
The process of plant breeding involving man's intervention in natural breeding and selection is some 20,000 years old. It has produced remarkable advances in adapting existing species to serve new purposes. The world's economics was largely based on the successes of agriculture for most of these 20,000 years.
Plant breeding involves choosing parents, making crosses to allow recombination of gene (alleles) and searching for and selecting improved forms. Success depends on the genes/alleles available, the combinations required and the ability to create and find the correct combinations necessary to give the desired properties to the plant. Molecular genetics technologies are now capable of providing new genes, new alleles and the means of creating and selecting plants with the new, desired characteristics.
Plants specifically improved for agriculture, horticulture and other industries can be obtained using molecular technologies. As an example, great agronomic value can result from modulating the size of a plant as a whole or of any of its organs. The green revolution came about as a result of creating dwarf wheat plants, which produced a higher seed yield than taller plants because they could withstand higher levels and inputs of fertilizer and water.
Similarly, modulation of the size and stature of an entire plant, or a particular portion of a plant, allows production of plants better suited for a particular industry. For example, reductions in the height of specific ornamentals, crops and tree species can be beneficial by allowing easier harvesting. Alternatively, increasing height may be beneficial by providing more biomass. Other examples of commercially desirable traits include increasing the length of the floral stems of cut flowers, increasing or altering leaf size and shape, enhancing the size of seeds and/or fruits, enhancing yields by specifically stimulating hormone (e.g. Brassinolide) synthesis and stimulating early flowering or evoking late flowering by altering levels of gibberellic acid or other hormones in specific cells. Changes in organ size and biomass also result in changes in the mass of constituent molecules such as secondary products.
To summarize, molecular genetic technologies provide the ability to modulate and manipulate growth, development and biochemistry of the entire plant as well as at the cell, tissue and organ levels. Thus, plant morphology, development and biochemistry are altered to maximize or minimize the desired plant trait.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated polynucleotides, polypeptides encoded thereby, and the use of those products for making transgenic organisms, such as plants, bacteria, yeast, fungi and mammals, depending upon the desired characteristics.
In the field of agriculture and forestry efforts are constantly being made to produce plants with improved characteristics, such as increased overall yield or increased yield of biomass or chemical components, in particular in order to guarantee the supply of the constantly increasing world population with food and to guarantee the supply of reproducible raw materials, Conventionally, people try to obtain plants with an increased yield by breeding, but this is time-consuming and labor-intensive. Furthermore, appropriate breeding programs must be performed for each relevant plant species.
Recently, progress has been made by the genetic manipulation of plants. That is, by introducing into and expressing recombinant nucleic acid molecules in plants. Such approaches have the advantage of not usually being limited to one plant species, but being transferable to other plant species as well. EP-A 0 511 979, for example, discloses that the expression of a prokaryotic asparagine synthetase in plant cells inter alia leads to an increase in biomass production. Similarly, WO 96/21737 describes the production of plants with increased yield from the expression of deregulated or unregulated fructose-1,6-bisphosphatase due to an increased rate of the photosynthesis. Nevertheless, there still is a need for generally applicable processes that improve yield in plants interesting for agriculture or forestry purposes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows alignments of the polypeptide sequences of the invention with other sequences, showing conserved regions of identical or similar residues.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

The following terms are utilized throughout this application:
Chimeric: The term “chimeric” is used to describe genes, as defined supra, or contructs wherein at least two of the elements of the gene or construct, such as the promoter and the coding sequence and/or other regulatory sequences and/or filler sequences and/or complements thereof, are heterologous to each other.
Constitutive Promoter: Promoters referred to herein as “constitutive promoters” actively promote transcription under most, but not necessarily all, environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcript initiation region and the 1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes, such as the maize ubiquitin-1 promoter, known to those of skill.
Domain: Domains are fingerprints or signatures that can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three-dimensional conformation. Generally, each domain has been associated with either a family of proteins or motifs. Typically, these families and/or motifs have been correlated with specific in-vitro and/or in-vivo activities. A domain can be any length, including the entirety of the sequence of a protein. Detailed descriptions of the domains, associated families and motifs, and correlated activities of the polypeptides of the instant invention are described below. Usually, the polypeptides with designated domain(s) can exhibit at least one activity that is exhibited by any polypeptide that comprises the same domain(s). Domains also define areas of non-coding sequences such as promoters and miRNAs.
Endogenous: The term “endogenous,” within the context of the current invention refers to any polynucleotide, polypeptide or protein sequence which is a natural part of a cell or organisms regenerated from said cell.
Exogenous: “Exogenous,” as referred to within, is any polynucleotide, polypeptide or protein sequence, whether chimeric or not, that is initially or subsequently introduced into the genome of an individual host cell or the organism regenerated from said host cell by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation (of dicots—e.g. Salomon et al. EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983); of monocots, representative papers are those by Escudero et al., Plant J. 10:355 (1996), Ishida et al., Nature Biotechnology 14:745 (1996), May et al., Bio/Technology 13:486 (1995)), biolistic methods (Armaleo et al., Current Genetics 17:97 1990)), electroporation, in planta techniques, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T₀for the primary transgenic plant and T₁for the first generation. The term “exogenous” as used herein is also intended to encompass inserting a naturally found element into a non-naturally found location.
Gene: The term “gene,” as used in the context of the current invention, encompasses all regulatory and coding sequence contiguously associated with a single hereditary unit with a genetic function. Genes can include non-coding sequences that modulate the genetic function that include, but are not limited to, those that specify polyadenylation, transcriptional regulation, DNA conformation, chromatin conformation, extent and position of base methylation and binding sites of proteins that control all of these. Genes comprised of “exons” (coding sequences), which may be interrupted by “introns” (non-coding sequences), encode proteins. A gene's genetic function may require only RNA expression or protein production, or may only require binding of proteins and/or nucleic acids without associated expression. In certain cases, genes adjacent to one another may share sequence in such a way that one gene will overlap the other. A gene can be found within the genome of an organism, artificial chromosome, plasmid, vector, etc., or as a separate isolated entity.
Heterologous sequences: “Heterologous sequences” are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from corn is considered heterologous to an Arabidopsis coding region sequence. Also, a promoter from a gene encoding a growth factor from corn is considered heterologous to a sequence encoding the corn receptor for the growth factor. Regulatory element sequences, such as UTRs or 3′ end termination sequences that do not originate in nature from the same gene as the coding sequence originates from, are considered heterologous to said coding sequence. Elements operatively linked in nature and contiguous to each other are not heterologous to each other. On the other hand, these same elements remain operatively linked but become heterologous if other filler sequence is placed between them. Thus, the promoter and coding sequences of a corn gene expressing an amino acid transporter are not heterologous to each other, but the promoter and coding sequence of a corn gene operatively linked in a novel manner are heterologous.
Homologous gene: In the current invention, “homologous gene” refers to a gene that shares sequence similarity with the gene of interest. This similarity may be in only a fragment of the sequence and often represents a functional domain such as, examples including without limitation a DNA binding domain, a domain with tyrosine kinase activity, or the like. The functional activities of homologous genes are not necessarily the same.
Misexpression: The term “misexpression” refers to an increase or a decrease in the transcription of a coding region into a complementary RNA sequence as compared to the parental wild-type. This term also encompasses expression of a gene or coding region for a different time period as compared to the wild-type and/or from a non-natural location within the plant genome.
Percentage of sequence identity: “Percentage of sequence identity,” as used herein, is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) 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. Optimal alignment of 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 (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Given that 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. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90% and most preferably at least 95%, even more preferably, at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.
Regulatory Sequence The term “regulatory sequence,” as used in the current invention, refers to any nucleotide sequence that influences transcription or translation initiation and rate, and stability and/or mobility of the transcript or polypeptide product. Regulatory sequences include, but are not limited to, promoters, promoter control elements, protein binding sequences, 5′ and 3′ UTRs, transcriptional start site, termination sequence, polyadenylation sequence, introns, certain sequences within a coding sequence, etc.
Stringency: “Stringency” as used herein is a function of probe length, probe composition (G+C content), and salt concentration, organic solvent concentration, and temperature of hybridization or wash conditions. Stringency is typically compared by the parameter T_m, which is the temperature at which 50% of the complementary molecules in the hybridization are hybridized, in terms of a temperature differential from T_m. High stringency conditions are those providing a condition of T_m−5° C. to T_m−11° C. Medium or moderate stringency conditions are those providing T_m−20° C. to T_m−29° C. Low stringency conditions are those providing a condition of T_m−40° C. to T_m−48° C. The relationship of hybridization conditions to T_m(in ° C.) is expressed in the mathematical equation
T _m=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N) (1)
where N is the length of the probe. This equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence. The equation below for T_mof DNA-DNA hybrids is useful for probes in the range of 50 to greater than 500 nucleotides, and for conditions that include an organic solvent (formamide).
T _m=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(% formamide) (2)
where L is the length of the probe in the hybrid. (P. Tijessen, “Hybridization with Nucleic Acid Probes” in Laboratory Techniques in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam.) The T_mof equation (2) is affected by the nature of the hybrid; for DNA-RNA hybrids T_mis 10-15° C. higher than calculated, for RNA-RNA hybrids T_mis 20-25° C. higher. Because the T_mdecreases about 1° C. for each 1% decrease in homology when a long probe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)), stringency conditions in polynucleotide hybridization reactions can be adjusted to favor hybridization of polynucleotides from identical genes or related family members.
Equation (2) is derived assuming equilibrium and therefore, hybridizations according to the present invention are most preferably performed under conditions of probe excess and for sufficient time to achieve equilibrium. The time required to reach equilibrium can be shortened by inclusion of a hybridization accelerator such as dextran sulfate or another high volume polymer in the hybridization buffer.
Stringency conditions can be selected during the hybridization reaction or after hybridization has occurred by altering the salt and temperature conditions of the wash solutions used. The formulas shown above are equally valid when used to compute the stringency of a wash solution. Preferred wash solution stringencies lie within the ranges stated above; high stringency is 5-8° C. below T_m, medium or moderate stringency is 26-29° C. below T_mand low stringency is 45-48° C. below T_m.
Substantially free of: A composition containing A is “substantially free of” B when at least 85% by weight of the total A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95% or even 99% by weight. For example, a plant gene or DNA sequence can be considered substantially free of other plant genes or DNA sequences.
Translational start site: In the context of the current invention, a “translational start site” is usually an ATG in the cDNA transcript, more usually the first ATG. A single cDNA, however, may have multiple translational start sites.
Transcription start site: “Transcription start site” is used in the current invention to describe the point at which transcription is initiated. This point is typically located about 25 nucleotides downstream from a TFIID binding site, such as a TATA box. Transcription can initiate at one or more sites within the gene, and a single gene may have multiple transcriptional start sites, some of which may be specific for transcription in a particular cell-type or tissue.
Untranslated region (UTR): A “UTR” is any contiguous series of nucleotide bases that is transcribed, but is not translated. These untranslated regions may be associated with particular functions such as increasing mRNA message stability. Examples of UTRs include, but are not limited to polyadenylation signals, terminations sequences, sequences located between the transcriptional start site and the first exon (5′ UTR) and sequences located between the last exon and the end of the mRNA (3′ UTR).
Variant: The term “variant” is used herein to denote a polypeptide or protein or polynucleotide molecule that differs from others of its kind in some way. For example, polypeptide and protein variants can consist of changes in amino acid sequence and/or charge and/or post-translational modifications (such as glycosylation, etc).

2. Important Characteristics of the Polynucleotides of the Invention

The genes and polynucleotides of the present invention are of interest because when they are misexpressed (i.e. when expressed at a non-natural location or in an increased amount) they produce plants with important modified characteristics as discussed below. These traits can be used to exploit or maximize plant products or to minimize undesirable characteristics. For example, an increase in plant height is beneficial in species grown or harvested for their main stem or trunk, such as ornamental cut flowers, fiber crops (e.g. flax, kenaf, hesperaloe, hemp) and wood producing trees. Increase in inflorescence thickness is also desirable for some ornamentals, while increases in the number, shape and size of leaves can lead to increased production/harvest from leaf crops such as lettuce, spinach, cabbage and tobacco. Likewise, a decrease in plant height is beneficial in species that are particularly susceptible to lodging or uprooting due to wind stress.
The polynucleotides and polypeptides of the invention were isolated from Arabidopsis thaliana, corn, soybean, wheat, Brassica and others as noted in the Sequence Listing. The polynucleotides and polypeptides are useful to confer on transgenic plants the properties identified for each sequence in the relevant portion (miscellaneous feature section) of the Sequence Listing. The miscellaneous feature section of the sequence listing contains, for each sequence, a description of the domain or other characteristic from which the sequence has the function known in the art for other sequences. Some identified domains are indicated with “PFam Name”, signifying that the pfam name and description can be found in the pfam database at http://pfam.wustl.edu. Other domains are indicated by reference to a “GI Number” from the public sequence database maintained by GenBank under the NCBI, including the non-redundant (NR) database.
The sequences of the invention can be applied to substrates for use in array applications such as, but not limited to, assays of global gene expression, under varying conditions of development, and growth conditions. The arrays are also used in diagnostic or forensic methods
The polynucleotides, or fragments thereof, can also be used as probes and primers. Probe length varies depending on the application. For use as primers, probes are 12-40 nucleotides, preferably 18-30 nucleotides long. For use in mapping, probes are preferably 50 to 500 nucleotides, preferably 100-250 nucleotides long. For Southern hybridizations, probes as long as several kilobases are used.
The probes and/or primers are produced by synthetic procedures such as 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) or using commercially available automated oligonucleotide synthesizers.
The polynucleotides of the invention can be utilized in a number of methods known to those skilled in the art as probes and/or primers to isolate and detect polynucleotides including, without limitation: Southerns, Northerns, Branched DNA hybridization assays, polymerase chain reaction microarray assays and variations thereof. Specific methods given by way of examples, and discussed below include:
hybridization
Methods of Mapping
Southern Blotting
Isolating cDNA from Related Organisms
Isolating and/or Identifying Homologous and Orthologous Genes.
Also, the nucleic acid molecules of the invention can be used in other methods, such as high density oligonucleotide hybridizing assays, described, for example, in U.S. Pat. Nos. 6,004,753 and 5,945,306.
The polynucleotides or fragments thereof of the present invention can be used as probes and/or primers for detection and/or isolation of related polynucleotide sequences through hybridization. Hybridization of one nucleic acid to another constitutes a physical property that defines the polynucleotide of the invention and the identified related sequences. Also, such hybridization imposes structural limitations on the pair. A good general discussion of the factors for determining hybridization conditions is provided by Sambrook et al. (“Molecular Cloning, a Laboratory Manual, 2nd ed., c. 1989 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; see esp., chapters 11 and 12). Additional considerations and details of the physical chemistry of hybridization are provided by G. H. Keller and M. M. Manak “DNA Probes”, 2nd Ed. pp. 1-25, c. 1993 by Stockton Press, New York, N.Y.
When using the polynucleotides to identify orthologous genes in other species, 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 are 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 erroneously being interpreted as absence of the corresponding gene in the genome.
The probes and/or primers of the instant invention can also be used to detect or isolate nucleotides that are “identical” to the probes or primers. Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
Isolated polynucleotides within the scope of the invention also include allelic variants of the specific sequences presented in the Sequence Listing. The probes and/or primers of the invention are also used to detect and/or isolate polynucleotides exhibiting at least 80% sequence identity with the sequences of the Sequence Listing or fragments thereof. Related polynucleotide sequences can also be identified according to the methods described in U.S. Patent Publication 20040137466A1, dated Jul. 15, 2004 to Jofuku et al.
With respect to nucleotide sequences, degeneracy of the genetic code provides the possibility to substitute at least one nucleotide of the nucleotide sequence of a gene with a different nucleotide without changing the amino acid sequence of the polypeptide. Hence, the DNA of the present invention also has any base sequence that has been changed from a sequence in the Sequence Listing by substitution in accordance with degeneracy of genetic code. References describing codon usage include: Carels et al., J. Mol. Evol. 46: 45 (1998) and Fennoy et al., Nucl. Acids Res. 21(23): 5294 (1993).
The polynucleotides of the invention are also used to create various types of genetic and physical maps of the genome of corn, Arabidopsis, soybean, rice, wheat, or other plants. Some are absolutely associated with particular phenotypic traits, allowing construction of gross genetic maps. Creation of such maps is based on differences or variants, generally referred to as polymorphisms, between different parents used in crosses. Common methods of detecting polymorphisms that can be used are restriction fragment length polymorphisms (RFLPs, single nucleotide polymorphisms (SNPs) or simple sequence repeats (SSRs).
The use of RFLPs and of recombinant inbred lines for such genetic mapping is described for Arabidopsis by Alonso-Blanco et al. (Methods in Molecular Biology, vol. 82, “Arabidopsis Protocols”, pp. 137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa, N.J.) and for corn by Burr (“Mapping Genes with Recombinant Inbreds”, pp. 249-254. In Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c. 1994 by Springer-Verlag New York, Inc.: New York, N.Y., USA; Berlin Germany; Burr et al. Genetics (1998) 118: 519; Gardiner, J. et al., (1993) Genetics 134: 917). This procedure, however, is not limited to plants and is used for other organisms (such as yeast) or for individual cells.
The polynucleotides of the present invention are also used for simple sequence repeat (SSR) mapping. Rice SSR mapping is described by Morgante et al. (The Plant Journal (1993) 3: 165), Panaud et al. (Genome (1995) 38: 1170); Senior et al. (Crop Science (1996) 36: 1676), Taramino et al. (Genome (1996) 39: 277) and Ahn et al. (Molecular and General Genetics (1993) 241: 483-90). SSR mapping is achieved using various methods. In one instance, polymorphisms are identified when sequence specific probes contained within a polynucleotide flanking an SSR are made and used in polymerase chain reaction (PCR) assays with template DNA from two or more individuals of interest. Here, a change in the number of tandem repeats between the SSR-flanking sequences produces differently sized fragments (U.S. Pat. No. 5,766,847). Alternatively, polymorphisms are identified by using the PCR fragment produced from the SSR-flanking sequence specific primer reaction as a probe against Southern blots representing different individuals (U. H. Refseth et al., (1997) Electrophoresis 18: 1519).
The polynucleotides of the invention can further be used to identify certain genes or genetic traits using, for example, known AFLP technologies, such as in EP0534858 and U.S. Pat. No. 5,878,215.
The polynucleotides of the present invention are also used for single nucleotide polymorphism (SNP) mapping.
Genetic and physical maps of crop species have many uses. For example, these maps are used to devise positional cloning strategies for isolating novel genes from the mapped crop species. In addition, because the genomes of closely related species are largely syntenic (i.e. they display the same ordering of genes within the genome), these maps are used to isolate novel alleles from relatives of crop species by positional cloning strategies.
The various types of maps discussed above are used with the polynucleotides of the invention to identify Quantitative Trait Loci (QTLs). Many important crop traits, such as the solids content of tomatoes, are quantitative traits and result from the combined interactions of several genes. These genes reside at different loci in the genome, often times on different chromosomes, and generally exhibit multiple alleles at each locus. The polynucleotides of the invention are used to identify QTLs and isolate specific alleles as described by de Vicente and Tanksley (Genetics 134:585 (1993)). Once a desired allele combination is identified, crop improvement is accomplished either through biotechnological means or by directed conventional breeding programs (for review see Tanksley and McCouch, Science 277:1063 (1997)). In addition to isolating QTL alleles in present crop species, the polynucleotides of the invention are also used to isolate alleles from the corresponding QTL of wild relatives.
In another embodiment, the polynucleotides are used to help create physical maps of the genome of corn, Arabidopsis and related species. Where polynucleotides are ordered on a genetic map, as described above, they are used as probes to discover which clones in large libraries of plant DNA fragments in YACs, BACs, etc. contain the same polynucleotide or similar sequences, thereby facilitating the assignment of the large DNA fragments to chromosomal positions. Subsequently, the large BACs, YACs, etc. are ordered unambiguously by more detailed studies of their sequence composition (e.g. Marra et al. (1997) Genomic Research 7:1072-1084) and by using their end or other sequences to find the identical sequences in other cloned DNA fragments. The overlapping of DNA sequences in this way allows building large contigs of plant sequences to be built that, when sufficiently extended, provide a complete physical map of a chromosome. Sometimes the polynucleotides themselves provide the means of joining cloned sequences into a contig. All scientific and patent publications cited in this paragraph are hereby incorporated by reference.
U.S. Pat. Nos. 6,287,778 and 6,500,614, both hereby incorporated by reference, describe scanning multiple alleles of a plurality of loci using hybridization to arrays of oligonucleotides. These techniques are useful for each of the types of mapping discussed above.
Following the procedures described above and using a plurality of the polynucleotides of the present invention, any individual is genotyped. These individual genotypes are used for the identification of particular cultivars, varieties, lines, ecotypes and genetically modified plants or can serve as tools for subsequent genetic studies involving multiple phenotypic traits.
Identification and isolation of orthologous genes from closely related species and alleles within a species is particularly desirable because of their potential for crop improvement. Many important crop traits, result from the combined interactions of the products of several genes residing at different loci in the genome. Generally, alleles at each of these loci make quantitative differences to the trait. Once a more favorable allele combination is identified, crop improvement is accomplished either through biotechnological means or by directed conventional breeding programs (Tanksley et al. Science 277:1063 (1997)).
FIG. 1 provides the results of ortholog analysis according to the invention. This analysis provides a means for identifying one or more sequences that are similar or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the method, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.
In particular, the polypeptide sequences of the invention (the “query sequences”) were used to query against the Applicant's own internal database of various plant sequences and against the entire NCBI GenBank database. This search resulted in an alignment for each query sequence with it's identified orthologous sequences, and that group of sequences provided the basis for identifying a respective consensus sequence. FIG. 1 sets forth the various alignments, wherein each query sequence is identified as a “Lead-Ceres Clone” followed by a numerical ID, the orthologs identified from the Applicant's internal database are identified as “Ceres Clone” followed by a numerical ID, and the orthologs identified from GenBank are identified as “gi” followed by a numerical ID. Each ortholog group, consisting of a query sequence (Lead-Ceres Clone), the identified orthologs, and the respective consensus sequence begins with a title that includes an identification of the relevant Lead-Ceres Clone.
The alignments of FIG. 1 also include an identification of the conserved domains or conserved regions, namely those domains or regions that are conserved across the group of orthologous sequences. One skilled in the art will recognize that each of the sequences in a particular ortholog group will be useful for the same purpose(s) as the Lead-Ceres Clone of that group, and that other useful orthologs can be designed or identified by taking into consideration the conserved regions or domains.
To aid in understanding the relationship of the various sequence identifiers used in this application, Table 1 provides a cross-reference for each polynucleotide sequence. In particular, Table 1 matches each polynucleotide sequence in the Sequence Listing (“SEQ ID NO:”) with (1) a number referred to as the “Ceres Clone ID” that is cited in the Sequence Listing as an internal identifier for the Applicant; (2) a similar identifier also utilized in the Sequence Listing and (3) the identifier utilized in the Homolog Table of FIG. 1 that references the sequence as a “Lead-Ceres Clone” and was used as the query sequence for identifying a homologous group of sequences. The SEQ ID NOS. in the Sequence Listing that are not in the Table 1 cross reference represent the polypeptide sequences that are coded by the next prior SEQ ID NO. or are part of the homolog group identified in FIG. 1. For example, SEQ ID NO:1 is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO:2 while SEQ ID NOS: 3-4 are the homologs of SEQ ID NO: 2 as shown in FIG. 1. Similarly, SEQ ID NO:5 is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO:6 while SEQ ID NOS: 7-21 are the homologs of SEQ ID NO: 6 as shown in FIG. 1.

4. Use of the Genes to Make Transgenic Plants

To use the sequences of the present invention or a combination of them or parts and/or mutants and/or fusions and/or variants of them, recombinant DNA constructs are prepared which comprise the polynucleotide sequences of the invention inserted into a vector, and which are suitable for transformation of plant cells. The construct is made using standard recombinant DNA techniques (Sambrook et al. 1989) and is introduced to the species of interest by Agrobacterium-mediated transformation or by other means of transformation as referenced below.
The vector backbone is any of those typical in the art such as plasmids (such as Ti plasmids), viruses, artificial chromosomes, BACs, YACs and PACs and vectors of the sort described by

(a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797 (1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979 (1996);
(b) YAC: Burke et al., Science 236:806-812 (1987);
(c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January; 87(1): 103-7 (1990);
(d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res 23: 4850-4856 (1995);
(e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et al., J. Mol. Biol 170: 827-842 (1983); or Insertion vector, e.g., Huynh et al., In: Glover N M (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press (1985); T-DNA gene fusion vectors Walden et al., Mol Cell Biol 1: 175-194 (1990); and
(g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a sequence of the present invention with any desired transcriptional and/or translational regulatory sequences, such as promoters, UTRs, and 3′ end termination sequences. Vectors can also include origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, introns, etc. The vector may also comprise a marker gene that confers a selectable phenotype on plant cells. The marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron, glyphosate or phosphinotricin.
A plant promoter fragment is used that directs transcription of the gene in all tissues of a regenerated plant and/or is a constitutive promoter. Alternatively, the plant promoter directs transcription of a sequence of the invention in a specific tissue (tissue-specific promoter) or is otherwise under more precise environmental control (inducible promoter).
If proper polypeptide production is desired, a polyadenylation region at the 3′-end of the coding region is typically included. The polyadenylation region is derived from the natural gene, from a variety of other plant genes, or from T-DNA, synthesized in the laboratory.

Transformation

Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g. Weising et al., Ann. Rev. Genet. 22:421 (1988); and Christou, Euphytica, v. 85, n. 1-3:13-27, (1995).
The person skilled in the art knows processes for the transformation of monocotyledonous and dicotyledonous plants. A variety of techniques are available for introducing DNA into a plant host cell. These techniques comprise transformation of plant cells by DNA injection, DNA electroporation, use of bolistics methods, protoplast fusion and via T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, as well as further possibilities, or other bacterial hosts for Ti plasmid vectors. See for example, Broothaerts et al., Gene Transfer to Plants by Diverse Species of Bacteria, Nature, Vol. 433, pp. 629-633, 10 Feb. 2005.
DNA constructs of the invention are introduced into the cell or the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct is introduced using techniques such as electroporation, microinjection and polyethylene glycol precipitation of plant cell protoplasts or protoplast fusion. Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Microinjection techniques are known in the art and well described in the scientific and patent literature. The plasmids do not have to fulfill specific requirements for use in DNA electroporation or DNA injection into plant cells. Simple plasmids such as pUC derivatives can be used.
The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717 (1984). Introduction of foreign DNA using protoplast fusion is described by Willmitzer (Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).
Alternatively, the DNA constructs of the invention are introduced directly into plant tissue using ballistic methods, such as DNA particle bombardment. Ballistic transformation techniques are described in Klein et al. Nature 327:773 (1987). Introduction of foreign DNA using ballistics is described by Willmitzer (Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).
DNA constructs are also introduced with the help of Agrobacteria. The use of Agrobacteria for plant cell transformation is extensively examined and sufficiently disclosed in the specification of EP-A 120 516, and in Hoekema (In: The Binary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V), Fraley et al. (Crit. Rev. Plant. Sci, 4, 1-46) and DePicker et al. (EMBO J. 4 (1985), 277-287). Using this technique, the DNA constructs of the invention are combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host direct the insertion of the construct and adjacent marker(s) into the plant cell DNA when the cell is infected by the bacteria (McCormac et al., 1997, Mol. Biotechnol. 8:199; Hamilton, 1997, Gene 200:107; Salomon et al., 1984 EMBO J. 3:141; Herrera-Estrella et al., 1983 EMBO J. 2:987). Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary or co-integrate vectors, are well described in the scientific literature. See, for example Hamilton, CM., Gene 200:107 (1997); Müller et al. Mol. Gen. Genet. 207:171 (1987); Komari et al. Plant J. 10:165 (1996); Venkateswarlu et al. Biotechnology 9:1103 (1991) and Gleave, AP., Plant Mol. Biol. 20:1203 (1992); Graves and Goldman, Plant Mol. Biol. 7:34 (1986) and Gould et al., Plant Physiology 95:426 (1991).
For plant cell T-DNA transfer of DNA, plant organs, e.g. infloresences, plant explants, plant cells that have been cultured in suspension or protoplasts are co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes or other suitable T-DNA hosts. Whole plants are regenerated from the infected plant material or seeds generated from infected plant material using a suitable medium that contains antibiotics or biocides for the selection of transformed cells or by spraying the biocide on plants to select the transformed plants. Plants obtained in this way are then examined for the presence of the DNA introduced. The transformation of dicotyledonous plants via Ti-plasmid-vector systems and Agrobacterium tumefaciens is well established.
Monocotyledonous plants are also transformed by means of Agrobacterium based vectors (See Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282; Deng et al., Science in China 33 (1990), 28-34; Wilmink et al., Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492; Conner and Domisse; Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265). Maize transformation in particular is described in the literature (see, for example, WO95/06128, EP 0 513 849; EP 0 465 875; Fromm et al., Biotechnology 8 (1990), 833-844; Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200). In EP 292 435 and in Shillito et al. (1989, Bio/Technology 7, 581) fertile plants are obtained from a mucus-free, soft (friable) maize callus. Prioli and Söndahl (1989, Bio/Technology 7, 589) also report regenerating fertile plants from maize protoplasts of the maize Cateto inbred line, Cat 100-1.
Other cereal species have also been successfully transformed, such as barley (Wan and Lemaux, see above; Ritala et al., see above) and wheat (Nehra et al., 1994, Plant J. 5, 285-297).
Alternatives to Agrobacterium transformation for plants are ballistics, protoplast fusion, electroporation of partially permeabilized cells and use of glass fibers (>See Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631)).
Introduced DNA is usually stable after integration into the plant genome and is transmitted to the progeny of the transformed cell or plant. Generally the transformed plant cell contains a selectable marker that makes the transformed cells resistant to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, phosphinotricin or others. Therefore, the individually chosen marker should allow the selection of transformed cells from cells lacking the introduced DNA.
The transformed cells grow within the plant in the usual way (McCormick et al., 1986, Plant Cell Reports 5, 81-84) and the resulting plants are cultured normally. Transformed plant cells obtained by any of the above transformation techniques are cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences.
Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture in “Handbook of Plant Cell Culture,” pp. 124-176, MacMillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1988. Regeneration also occurs from plant callus, explants, organs, or pairs thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467 (1987). Regeneration of monocots (rice) is described by Hosoyama et al. (Biosci. Biotechnol. Biochem. 58:1500 (1994)) and by Ghosh et al. (J. Biotechnol. 32:1 (1994)). Useful and relevant procedures for transient expression are also described in U.S. Application No. 60/537,070 filed on Jan. 16, 2004 and PCT Application No. PCT/US2005/001153 filed on Jan. 14, 2005.
After transformation, seeds are obtained from the plants and used for testing stability and inheritance. Generally, two or more generations are cultivated to ensure that the phenotypic feature is stably maintained and transmitted.
One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
The nucleotide sequences according to the invention generally encode an appropriate protein from any organism, in particular from plants, fungi, bacteria or animals. The sequences preferably encode proteins from plants or fungi. Preferably, the plants are higher plants, in particular starch or oil storing useful plants, such as potato or cereals such as rice, maize, wheat, barley, rye, triticale, oat, millet, etc., as well as spinach, tobacco, sugar beet, soya, cotton etc.
In principle, the process according to the invention can be applied to any plant.
Therefore, monocotyledonous as well as dicotyledonous plant species are particularly suitable. The process is preferably used with plants that are interesting for agriculture, horticulture and/or forestry. Examples are vegetable plants such as cucumber, melon, pumpkin, eggplant, zucchini, tomato, spinach, cabbage species, peas, beans, etc., as well as fruits such as pears, apples, etc.
Thus, the invention has use over a broad range of plants, preferably higher plants, pertaining to the classes of Angiospermae and Gymnospermae. Plants of the subclasses of the Dicotylodenae and the Monocotyledonae are particularly suitable. Dicotyledonous plants belong to the orders of the Magniolales, Illiciales, Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecyihidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales. Monocotyledonous plants belong to the orders of the Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchidales. Plants belonging to the class of the Gymnospermae are Pinales, Ginkgoales, Cycadales and Gnetales.
The method of the invention is preferably used with plants that are interesting for agriculture, horticulture, biomass for bioconversion and/or forestry. Examples are tobacco, oilseed rape, sugar beet, potato, tomato, cucumber, pepper, bean, pea, citrus fruit, apple, pear, berries, plum, melon, eggplant, cotton, soybean, sunflower, rose, poinsettia, petunia, guayule, cabbage, spinach, alfalfa, artichoke, corn, wheat, rye, barley, grasses such as switch grass or turf grass, millet, hemp, banana, poplar, eucalyptus trees, conifers.

5. Phenotype Screens and Results

5.1 Triparental Mating and Vacuum Infiltration Transformation of Plants
The function/phenotype characteristics of the sequences of the invention were determined by means of screens with transgenic plants. Standard laboratory techniques are as described in Sambrook et al. (1989) unless otherwise stated. Single colonies of Agrobacterium C58C1Rif, E. coli helper strain HB101 and the E. coli strain containing the transformation construct to be mobilized into Agrobacterium are separately inoculated into appropriate growth media and stationary cultures produced. Cultures are mixed gently, plated on YEB (5 g Gibco beef extract, 1 g Bacto yeast extract, 1 g Bacto peptone, 5 g sucrose, pH 7.4) solid growth media and incubated overnight at 28° C. The bacteria from the triparental mating are collected in and serial dilutions made. An aliquot of the each dilution is then plated and incubated for 2 days at 28° C. on YEB plates supplemented with 100 μg/ml rifampicin and 100 μg/ml carbenicillin for calculation of the number of acceptor cells and on YEB plates supplemented with 100 μg/ml rifampicin, 100 μg/ml carbenicillin and 100 μg/ml spectinomycin for selection of transconjugant cells. The cointegrate structure of purified transconjugants is verified via Southern blot hybridization.
A transconjugant culture is prepared for vacuum infiltration by inoculating 1 ml of a stationary culture arising from a single colony into liquid YEB media and incubating at 28° C. for approximately 20 hours with shaking until the OD taken at 600 nm was 0.8-1.0. The culture is then pelleted and the bacteria resuspended in infiltration medium (0.5× MS salts, 5% w/v sucrose, 10 μg/l BAP, 200 μl/l Silwet L-77, pH 5.8) to a final OD₆₀₀of 1.0. This prepared transconjugant culture is used within 20 minutes of preparation.
Wild-type plants for vacuum infiltration are grown in pots. Briefly, seeds of Arabidopsis thaliana (ecotype Wassilewskija) are sown in pots and left at 4° C. for two to four days to vernalize. They are then transferred to 22-25° C. and grown under long-day (16 hr light: 8 hr dark) conditions, sub-irrigated with water. After bolting, the primary inflorescence is removed and, after four to eight days, the pots containing the plants are inverted in the vacuum chamber to submerge all of the plants in the prepared transconjugant culture. Vacuum is drawn for two minutes before pots are removed, covered with plastic wrap and incubated in a cool room under darkness or very low light for one to two days. The plastic wrap is then removed; the plants returned to their previous growing conditions and subsequently produced (T1) seed collected.
5.2 Selection of T-DNA Insertion Lines
Seeds from the initial vacuum infiltrated plants are sown on flats of Metromix 350 soil. Flats are vernalized for four to five days at 4° C. before being transferred to 22-25° C. and grown under long-day (16 hr light: 8 hr dark) conditions, sub-irrigated with water. Approximately seven to ten days after germination, the (T1) seedlings are sprayed with 0.02% Finale herbicide (AgrEvo). After another five to seven days, herbicide treatment is repeated. Herbicide resistant T1 plants are allowed to self-pollinate and T2 seed are collected from each individual. In the few cases where the T1 plant produced few seed, the T2 seed is planted in bulk, the T2 plants allowed to self-pollinate and T3 seed collected.
5.3 Phenotype Screening
Seeds from each T2 (or T3) line are planted in a 4-inch pot containing either Sunshine mix or Metromix 350 soil. Pots are vernalized for four to five days at 4° C. before being transferred to 22-25° C. and grown under long-day (16 hr light: 8 hr dark) conditions, sub-irrigated with water. A first phenotype screen is conducted by visually inspecting the seedlings five to seven days after germination and aberrant phenotypes noted. Plants are then sprayed with Finale herbicide within four days (i.e. about seven to nine days after germination). The second visual screen is conducted on surviving T2 (or T3) plants about sixteen to seventeen days after germination and the final screen was conducted after the plants have bolted and formed siliques. Here, the third and fourth green siliques are collected and aberrant phenotypes noted.
Alternatively, seed are surface sterilized and transferred to agar solidified medium containing Murashige and Skoog salts (1×), 1% sucrose (wt/v) pH 5.7 before autoclaving. Seed re cold treated for 48 hours and transferred to long days [16 hours light and 8 hours dark], 25° C. Plants are screened at 5 and 10 days.
The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.
Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.

TABLE 1

SEQUECE IDENTIFIER CROSS-REFERENCE

	CERES
	CLONE
SEQ ID NO	ID	CERES ID IN SEQUENCE LISTING	HOMOLOG_TABLE_ID

SEQ ID NO: 1	40252	Identifier: Ceres CLONE ID no. 40252	Lead_CeresClone40252
SEQ ID NO: 5	32791	Identifier: Ceres CLONE ID no. 32791	Lead_CeresClone32791
SEQ ID NO: 22	39319	Identifier: Ceres CLONE ID no. 39319	Lead_CeresClone39319
SEQ ID NO: 37	41337	Identifier: Ceres CLONE ID no. 41337	Lead_CeresClone41337
SEQ ID NO: 46	314	Identifier: Ceres CLONE ID no. 314	Lead_CeresClone314
SEQ ID NO: 54	332	Identifier: Ceres CLONE ID no. 332	Lead_CeresClone332
SEQ ID NO: 66	907	Identifier: Ceres CLONE ID no. 907	Lead_CeresClone907
SEQ ID NO: 71	1241	Identifier: Ceres CLONE ID no. 1241	Lead_CeresClone1241
SEQ ID NO: 79	1610	Identifier: Ceres CLONE ID no. 1610	Lead_CeresClone1610
SEQ ID NO: 95	2403	Identifier: Ceres CLONE ID no. 2403	Lead_CeresClone2403
SEQ ID NO: 107	2835	Identifier: Ceres CLONE ID no. 2835	Lead_CeresClone2835
SEQ ID NO: 114	3000	Identifier: Ceres CLONE ID no. 3000	Lead_CeresClone3000
SEQ ID NO: 123	3036	Identifier: Ceres CLONE ID no. 3036	Lead_CeresClone3036
SEQ ID NO: 128	3363	Identifier: Ceres CLONE ID no. 3363	Lead_CeresClone3363
SEQ ID NO: 147	3510	Identifier: Ceres CLONE ID no. 3510	Lead_CeresClone3510
SEQ ID NO: 161	3699	Identifier: Ceres CLONE ID no. 3699	Lead_CeresClone3699
SEQ ID NO: 176	3858	Identifier: Ceres CLONE ID no. 3858	Lead_CeresClone3858
SEQ ID NO: 191	5597	Identifier: Ceres CLONE ID no. 5597	Lead_CeresClone5597
SEQ ID NO: 199	5605	Identifier: Ceres CLONE ID no. 5605	Lead_CeresClone5605
SEQ ID NO: 210	6685	Identifier: Ceres CLONE ID no. 6685	Lead_CeresClone6685
SEQ ID NO: 215	8265	Identifier: Ceres CLONE ID no. 8265	Lead_CeresClone8265
SEQ ID NO: 221	8490	Identifier: Ceres CLONE ID no. 8490	Lead_CeresClone8490
SEQ ID NO: 230	9683	Identifier: Ceres CLONE ID no. 9683	Lead_CeresClone9683
SEQ ID NO: 234	9897	Identifier: Ceres CLONE ID no. 9897	Lead_CeresClone9897
SEQ ID NO: 242	12272	Identifier: Ceres CLONE ID no. 12272	Lead_CeresClone12272
SEQ ID NO: 251	12935	Identifier: Ceres CLONE ID no. 12935	Lead_CeresClone12935
SEQ ID NO: 262	13092	Identifier: Ceres CLONE ID no. 13092	Lead_CeresClone13092
SEQ ID NO: 270	13263	Identifier: Ceres CLONE ID no. 13263	Lead_CeresClone13263
SEQ ID NO: 276	14583	Identifier: Ceres CLONE ID no. 14583	Lead_CeresClone14583
SEQ ID NO: 283	14909	Identifier: Ceres CLONE ID no. 14909	Lead_CeresClone14909
SEQ ID NO: 295	16412	Identifier: Ceres CLONE ID no. 16412	Lead_CeresClone16412
SEQ ID NO: 304	16461	Identifier: Ceres CLONE ID no. 16461	Lead_CeresClone16461
SEQ ID NO: 309	17409	Identifier: Ceres CLONE ID no. 17409	Lead_CeresClone17409
SEQ ID NO: 316	17482	Identifier: Ceres CLONE ID no. 17482	Lead_CeresClone17482
SEQ ID NO: 321	17632	Identifier: Ceres CLONE ID no. 17632	Lead_CeresClone17632
SEQ ID NO: 328	18612	Identifier: Ceres CLONE ID no. 18612	Lead_CeresClone18612
SEQ ID NO: 335	18820	Identifier: Ceres CLONE ID no. 18820	Lead_CeresClone18820
SEQ ID NO: 341	19188	Identifier: Ceres CLONE ID no. 19188	Lead_CeresClone19188
SEQ ID NO: 351	20257	Identifier: Ceres CLONE ID no. 20257	Lead_CeresClone20257
SEQ ID NO: 362	21068	Identifier: Ceres CLONE ID no. 21068	Lead_CeresClone21068
SEQ ID NO: 367	22461	Identifier: Ceres CLONE ID no. 22461	Lead_CeresClone22461
SEQ ID NO: 372	23203	Identifier: Ceres CLONE ID no. 23203	Lead_CeresClone23203
SEQ ID NO: 382	26907	Identifier: Ceres CLONE ID no. 26907	Lead_CeresClone26907
SEQ ID NO: 393	27460	Identifier: Ceres CLONE ID no. 27460	Lead_CeresClone27460
SEQ ID NO: 396	32348	Identifier: Ceres CLONE ID no. 32348	Lead_CeresClone32348
SEQ ID NO: 407	32548	Identifier: Ceres CLONE ID no. 32548	Lead_CeresClone32548
SEQ ID NO: 417	32753	Identifier: Ceres CLONE ID no. 32753	Lead_CeresClone32753
SEQ ID NO: 422	34167	Identifier: Ceres CLONE ID no. 34167	Lead_CeresClone34167
SEQ ID NO: 430	34385	Identifier: Ceres CLONE ID no. 34385	Lead_CeresClone34385
SEQ ID NO: 449	36518	Identifier: Ceres CLONE ID no. 36518	Lead_CeresClone36518
SEQ ID NO: 456	36891	Identifier: Ceres CLONE ID no. 36891	Lead_CeresClone36891
SEQ ID NO: 471	36904	Identifier: Ceres CLONE ID no. 36904	Lead_CeresClone36904
SEQ ID NO: 478	37288	Identifier: Ceres CLONE ID no. 37288	Lead_CeresClone37288
SEQ ID NO: 484	37298	Identifier: Ceres CLONE ID no. 37298	Lead_CeresClone37298
SEQ ID NO: 495	37663	Identifier: Ceres CLONE ID no. 37663	Lead_CeresClone37663
SEQ ID NO: 498	38101	Identifier: Ceres CLONE ID no. 38101	Lead_CeresClone38101
SEQ ID NO: 511	38419	Identifier: Ceres CLONE ID no. 38419	Lead_CeresClone38419
SEQ ID NO: 518	38470	Identifier: Ceres CLONE ID no. 38470	Lead_CeresClone38470
SEQ ID NO: 526	38690	Identifier: Ceres CLONE ID no. 38690	Lead_CeresClone38690
SEQ ID NO: 530	39286	Identifier: Ceres CLONE ID no. 39286	Lead_CeresClone39286
SEQ ID NO: 536	40508	Identifier: Ceres CLONE ID no. 40508	Lead_CeresClone40508
SEQ ID NO: 543	40729	Identifier: Ceres CLONE ID no. 40729	Lead_CeresClone40729
SEQ ID NO: 551	41306	Identifier: Ceres CLONE ID no. 41306	Lead_CeresClone41306
SEQ ID NO: 554	41439	Identifier: Ceres CLONE ID no. 41439	Lead_CeresClone41439
SEQ ID NO: 569	42141	Identifier: Ceres CLONE ID no. 42141	Lead_CeresClone42141
SEQ ID NO: 578	92459	Identifier: Ceres CLONE ID no. 92459	Lead_CeresClone92459
SEQ ID NO: 585	92670	Identifier: Ceres CLONE ID no. 92670	Lead_CeresClone92670
SEQ ID NO: 598	94231	Identifier: Ceres CLONE ID no. 94231	Lead_CeresClone94231
SEQ ID NO: 611	95135	Identifier: Ceres CLONE ID no. 95135	Lead_CeresClone95135
SEQ ID NO: 623	97434	Identifier: Ceres CLONE ID no. 97434	Lead_CeresClone97434
SEQ ID NO: 640	97480	Identifier: Ceres CLONE ID no. 97480	Lead_CeresClone97480
SEQ ID NO: 654	97958	Identifier: Ceres CLONE ID no. 97958	Lead_CeresClone97958
SEQ ID NO: 672	98855	Identifier: Ceres CLONE ID no. 98855	Lead_CeresClone98855
SEQ ID NO: 681	99657	Identifier: Ceres CLONE ID no. 99657	Lead_CeresClone99657
SEQ ID NO: 699	100465	Identifier: Ceres CLONE ID no. 100465	Lead_CeresClone100465
SEQ ID NO: 703	107731	Identifier: Ceres CLONE ID no. 107731	Lead_CeresClone107731
SEQ ID NO: 717	110454	Identifier: Ceres CLONE ID no. 110454	Lead_CeresClone110454
SEQ ID NO: 733	116843	Identifier: Ceres CLONE ID no. 116843	Lead_CeresClone116843
SEQ ID NO: 738	119256	Identifier: Ceres CLONE ID no. 119256	Lead_CeresClone119256
SEQ ID NO: 746	123905	Identifier: Ceres CLONE ID no. 123905	Lead_CeresClone123905
SEQ ID NO: 753	141805	Identifier: Ceres CLONE ID no. 141805	Lead_CeresClone141805
SEQ ID NO: 759	141890	Identifier: Ceres CLONE ID no. 141890	Lead_CeresClone141890
SEQ ID NO: 762	147358	Identifier: Ceres CLONE ID no. 147358	Lead_CeresClone147358
SEQ ID NO: 775	148943	Identifier: Ceres CLONE ID no. 148943	Lead_CeresClone148943
SEQ ID NO: 785	157547	Identifier: Ceres CLONE ID no. 157547	Lead_CeresClone157547
SEQ ID NO: 791	158333	Identifier: Ceres CLONE ID no. 158333	Lead_CeresClone158333
SEQ ID NO: 797	227651	Identifier: Ceres CLONE ID no. 227651	Lead_CeresClone227651
SEQ ID NO: 815	235672	Identifier: Ceres CLONE ID no. 235672	Lead_CeresClone235672
SEQ ID NO: 819	241131	Identifier: Ceres CLONE ID no. 241131	Lead_CeresClone241131
SEQ ID NO: 825	262460	Identifier: Ceres CLONE ID no. 262460	Lead_CeresClone262460
SEQ ID NO: 832	270555	Identifier: Ceres CLONE ID no. 270555	Lead_CeresClone270555
SEQ ID NO: 835	481710	Identifier: Ceres CLONE ID no. 481710	Lead_CeresClone481710
SEQ ID NO: 846	482122	Identifier: Ceres CLONE ID no. 482122	Lead_CeresClone482122
SEQ ID NO: 860	536457	Identifier: Ceres CLONE ID no. 536457	Lead_CeresClone536457
SEQ ID NO: 871	536796	Identifier: Ceres CLONE ID no. 536796	Lead_CeresClone536796
SEQ ID NO: 877	572121	Identifier: Ceres CLONE ID no. 572121	Lead_CeresClone572121
SEQ ID NO: 880	641355	Identifier: Ceres CLONE ID no. 641355	Lead_CeresClone641355
SEQ ID NO: 894	660003	Identifier: Ceres CLONE ID no. 660003	Lead_CeresClone660003
SEQ ID NO: 898	664365	Identifier: Ceres CLONE ID no. 664365	Lead_CeresClone664365
SEQ ID NO: 908	708342	Identifier: Ceres CLONE ID no. 708342	Lead_CeresClone708342
SEQ ID NO: 914	969750	Identifier: Ceres CLONE ID no. 969750	Lead_CeresClone969750
SEQ ID NO: 919	1001432	Identifier: Ceres CLONE ID no. 1001432	Lead_CeresClone1001432
SEQ ID NO: 930	1002819	Identifier: Ceres CLONE ID no. 1002819	Lead_CeresClone1002819
SEQ ID NO: 935	1007549	Identifier: Ceres CLONE ID no. 1007549	Lead_CeresClone1007549
SEQ ID NO: 948	1043081	Identifier: Ceres CLONE ID no. 1043081	Lead_CeresClone1043081
SEQ ID NO: 965	99298	Identifier: Ceres CLONE ID no. 99298	Lead_CeresClone99298
SEQ ID NO: 974	100245	Identifier: Ceres CLONE ID no. 100245	Lead_CeresClone100245
SEQ ID NO: 989	101798	Identifier: Ceres CLONE ID no. 101798	Lead_CeresClone101798
SEQ ID NO: 1005	38370	Identifier: Ceres CLONE ID no. 38370	Lead_CeresClone38370
SEQ ID NO: 1012	1496	Identifier: Ceres CLONE ID no. 1496	Lead_CeresClone1496
SEQ ID NO: 1031	2561	Identifier: Ceres CLONE ID no. 2561	Lead_CeresClone2561
SEQ ID NO: 1042	3618	Identifier: Ceres CLONE ID no. 3618	Lead_CeresClone3618
SEQ ID NO: 1048	7191	Identifier: Ceres CLONE ID no. 7191	Lead_CeresClone7191
SEQ ID NO: 1057	8254	Identifier: Ceres CLONE ID no. 8254	Lead_CeresClone8254
SEQ ID NO: 1062	8877	Identifier: Ceres CLONE ID no. 8877	Lead_CeresClone8877
SEQ ID NO: 1075	8916	Identifier: Ceres CLONE ID no. 8916	Lead_CeresClone8916
SEQ ID NO: 1078	10879	Identifier: Ceres CLONE ID no. 10879	Lead_CeresClone10879
SEQ ID NO: 1086	19116	Identifier: Ceres CLONE ID no. 19116	Lead_CeresClone19116
SEQ ID NO: 1092	19319	Identifier: Ceres CLONE ID no. 19319	Lead_CeresClone19319
SEQ ID NO: 1100	19486	Identifier: Ceres CLONE ID no. 19486	Lead_CeresClone19486
SEQ ID NO: 1106	19510	Identifier: Ceres CLONE ID no. 19510	Lead_CeresClone19510
SEQ ID NO: 1125	23322	Identifier: Ceres CLONE ID no. 23322	Lead_CeresClone23322
SEQ ID NO: 1136	25538	Identifier: Ceres CLONE ID no. 25538	Lead_CeresClone25538
SEQ ID NO: 1145	25607	Identifier: Ceres CLONE ID no. 25607	Lead_CeresClone25607
SEQ ID NO: 1155	25758	Identifier: Ceres CLONE ID no. 25758	Lead_CeresClone25758
SEQ ID NO: 1162	25886	Identifier: Ceres CLONE ID no. 25886	Lead_CeresClone25886
SEQ ID NO: 1181	27464	Identifier: Ceres CLONE ID no. 27464	Lead_CeresClone27464
SEQ ID NO: 1190	28602	Identifier: Ceres CLONE ID no. 28602	Lead_CeresClone28602
SEQ ID NO: 1204	35493	Identifier: Ceres CLONE ID no. 35493	Lead_CeresClone35493
SEQ ID NO: 1217	37229	Identifier: Ceres CLONE ID no. 37229	Lead_CeresClone37229
SEQ ID NO: 1227	37493	Identifier: Ceres CLONE ID no. 37493	Lead_CeresClone37493
SEQ ID NO: 1235	38105	Identifier: Ceres CLONE ID no. 38105	Lead_CeresClone38105
SEQ ID NO: 1242	38214	Identifier: Ceres CLONE ID no. 38214	Lead_CeresClone38214
SEQ ID NO: 1252	41320	Identifier: Ceres CLONE ID no. 41320	Lead_CeresClone41320
SEQ ID NO: 1262	42533	Identifier: Ceres CLONE ID no. 42533	Lead_CeresClone42533
SEQ ID NO: 1271	42925	Identifier: Ceres CLONE ID no. 42925	Lead_CeresClone42925
SEQ ID NO: 1282	95453	Identifier: Ceres CLONE ID no. 95453	Lead_CeresClone95453
SEQ ID NO: 1295	96020	Identifier: Ceres CLONE ID no. 96020	Lead_CeresClone96020
SEQ ID NO: 1302	97415	Identifier: Ceres CLONE ID no. 97415	Lead_CeresClone97415
SEQ ID NO: 1313	101255	Identifier: Ceres CLONE ID no. 101255	Lead_CeresClone101255
SEQ ID NO: 1321	103581	Identifier: Ceres CLONE ID no. 103581	Lead_CeresClone103581
SEQ ID NO: 1336	109514	Identifier: Ceres CLONE ID no. 109514	Lead_CeresClone109514
SEQ ID NO: 1346	115946	Identifier: Ceres CLONE ID no. 115946	Lead_CeresClone115946
SEQ ID NO: 1351	115975	Identifier: Ceres CLONE ID no. 115975	Lead_CeresClone115975
SEQ ID NO: 1359	117369	Identifier: Ceres CLONE ID no. 117369	Lead_CeresClone117369
SEQ ID NO: 1370	118337	Identifier: Ceres CLONE ID no. 118337	Lead_CeresClone118337
SEQ ID NO: 1383	150912	Identifier: Ceres CLONE ID no. 150912	Lead_CeresClone150912
SEQ ID NO: 1389	152141	Identifier: Ceres CLONE ID no. 152141	Lead_CeresClone152141
SEQ ID NO: 1403	157730	Identifier: Ceres CLONE ID no. 157730	Lead_CeresClone157730
SEQ ID NO: 1411	225597	Identifier: Ceres CLONE ID no. 225597	Lead_CeresClone225597
SEQ ID NO: 1415	264705	Identifier: Ceres CLONE ID no. 264705	Lead_CeresClone264705
SEQ ID NO: 1436	627596	Identifier: Ceres CLONE ID no. 627596	Lead_CeresClone627596
SEQ ID NO: 1450	729085	Identifier: Ceres CLONE ID no. 729085	Lead_CeresClone729085
SEQ ID NO: 1463	1011386	Identifier: Ceres CLONE ID no. 1011386	Lead_CeresClone1011386
SEQ ID NO: 1467	6082	Identifier: Ceres CLONE ID no. 6082	Lead_CeresClone6082
SEQ ID NO: 1474	13812	Identifier: Ceres CLONE ID no. 13812	Lead_CeresClone13812
SEQ ID NO: 1479	32811	Identifier: Ceres CLONE ID no. 32811	Lead_CeresClone32811
SEQ ID NO: 1485	224062	Identifier: Ceres CLONE ID no. 224062	Lead_CeresClone224062
SEQ ID NO: 1494	254065	Identifier: Ceres CLONE ID no. 254065	Lead_CeresClone254065
SEQ ID NO: 1502	22339	Identifier: Ceres CLONE ID no. 22339	Lead_CeresClone22339
SEQ ID NO: 1516	99784	Identifier: Ceres CLONE ID no. 99784	Lead_CeresClone99784
SEQ ID NO: 1532	100319	Identifier: Ceres CLONE ID no. 100319	Lead_CeresClone100319
SEQ ID NO: 1539	124720	Identifier: Ceres CLONE ID no. 124720	Lead_CeresClone124720
SEQ ID NO: 1548	288251	Identifier: Ceres CLONE ID no. 288251	Lead_CeresClone288251
SEQ ID NO: 1555	8014	Identifier: Ceres CLONE ID no. 8014	Lead_CeresClone8014
SEQ ID NO: 1562	16204	Identifier: Ceres CLONE ID no. 16204	Lead_CeresClone16204
SEQ ID NO: 1573	101250	Identifier: Ceres CLONE ID no. 101250	Lead_CeresClone101250
SEQ ID NO: 1579	283597	Identifier: Ceres CLONE ID no. 283597	Lead_CeresClone283597
SEQ ID NO: 1586	292789	Identifier: Ceres CLONE ID no. 292789	Lead_CeresClone292789
SEQ ID NO: 1606	4289	Identifier: Ceres CLONE ID no. 4289	Lead_CeresClone4289
SEQ ID NO: 1610	7925	Identifier: Ceres CLONE ID no. 7925	Lead_CeresClone7925
SEQ ID NO: 1613	10857	Identifier: Ceres CLONE ID no. 10857	Lead_CeresClone10857
SEQ ID NO: 1619	19481	Identifier: Ceres CLONE ID no. 19481	Lead_CeresClone19481
SEQ ID NO: 1626	28979	Identifier: Ceres CLONE ID no. 28979	Lead_CeresClone28979
SEQ ID NO: 1638	113719	Identifier: Ceres CLONE ID no. 113719	Lead_CeresClone113719
SEQ ID NO: 1651	147593	Identifier: Ceres CLONE ID no. 147593	Lead_CeresClone147593
SEQ ID NO: 1661	150798	Identifier: Ceres CLONE ID no. 150798	Lead_CeresClone150798
SEQ ID NO: 1666	152076	Identifier: Ceres CLONE ID no. 152076	Lead_CeresClone152076
SEQ ID NO: 1673	154031	Identifier: Ceres CLONE ID no. 154031	Lead_CeresClone154031
SEQ ID NO: 1677	246416	Identifier: Ceres CLONE ID no. 246416	Lead_CeresClone246416
SEQ ID NO: 1696	949	Identifier: Ceres CLONE ID no. 949	Lead_CeresClone949
SEQ ID NO: 1710	2036	Identifier: Ceres CLONE ID no. 2036	Lead_CeresClone2036
SEQ ID NO: 1719	18857	Identifier: Ceres CLONE ID no. 18857	Lead_CeresClone18857
SEQ ID NO: 1728	23518	Identifier: Ceres CLONE ID no. 23518	Lead_CeresClone23518
SEQ ID NO: 1745	156655	Identifier: Ceres CLONE ID no. 156655	Lead_CeresClone156655
SEQ ID NO: 1750	2273	Identifier: Ceres CLONE ID no. 2273	Lead_CeresClone2273
SEQ ID NO: 1756	5198	Identifier: Ceres CLONE ID no. 5198	Lead_CeresClone5198
SEQ ID NO: 1765	13767	Identifier: Ceres CLONE ID no. 13767	Lead_CeresClone13767
SEQ ID NO: 1773	29150	Identifier: Ceres CLONE ID no. 29150	Lead_CeresClone29150
SEQ ID NO: 1777	34480	Identifier: Ceres CLONE ID no. 34480	Lead_CeresClone34480
SEQ ID NO: 1785	38625	Identifier: Ceres CLONE ID no. 38625	Lead_CeresClone38625
SEQ ID NO: 1791	39351	Identifier: Ceres CLONE ID no. 39351	Lead_CeresClone39351
SEQ ID NO: 1800	153053	Identifier: Ceres CLONE ID no. 153053	Lead_CeresClone153053
SEQ ID NO: 1805	159318	Identifier: Ceres CLONE ID no. 159318	Lead_CeresClone159318
SEQ ID NO: 1811	241379	Identifier: Ceres CLONE ID no. 241379	Lead_CeresClone241379
SEQ ID NO: 1822	5220	Identifier: Ceres CLONE ID no. 5220	Lead_CeresClone5220
SEQ ID NO: 1826	11214	Identifier: Ceres CLONE ID no. 11214	Lead_CeresClone11214
SEQ ID NO: 1841	563522	Identifier: Ceres CLONE ID no. 563522	Lead_CeresClone563522
SEQ ID NO: 1860	21563	Identifier: Ceres CLONE ID no. 21563	Lead_CeresClone21563
SEQ ID NO: 1868	6397	Identifier: Ceres CLONE ID no. 6397	Lead_CeresClone6397
SEQ ID NO: 1872	14555	Identifier: Ceres CLONE ID no. 14555	Lead_CeresClone14555
SEQ ID NO: 1882	4067	Identifier: Ceres CLONE ID no. 4067	Lead_CeresClone4067
SEQ ID NO: 1895	4734	Identifier: Ceres CLONE ID no. 4734	Lead_CeresClone4734
SEQ ID NO: 1903	28643	Identifier: Ceres CLONE ID no. 28643	Lead_CeresClone28643
SEQ ID NO: 1917	733804	Identifier: Ceres CLONE ID no. 733804	Lead_CeresClone733804
SEQ ID NO: 1929	9221	Identifier: Ceres CLONE ID no. 9221	Lead_CeresClone9221
SEQ ID NO: 1943	11929	Identifier: Ceres CLONE ID no. 11929	Lead_CeresClone11929
SEQ ID NO: 1954	12071	Identifier: Ceres CLONE ID no. 12071	Lead_CeresClone12071
SEQ ID NO: 1961	13625	Identifier: Ceres CLONE ID no. 13625	Lead_CeresClone13625
SEQ ID NO: 1971	16865	Identifier: Ceres CLONE ID no. 16865	Lead_CeresClone16865
SEQ ID NO: 1988	18246	Identifier: Ceres CLONE ID no. 18246	Lead_CeresClone18246
SEQ ID NO: 1994	31044	Identifier: Ceres CLONE ID no. 31044	Lead_CeresClone31044
SEQ ID NO: 2001	38635	Identifier: Ceres CLONE ID no. 38635	Lead_CeresClone38635
SEQ ID NO: 2008	39155	Identifier: Ceres CLONE ID no. 39155	Lead_CeresClone39155
SEQ ID NO: 2011	107988	Identifier: Ceres CLONE ID no. 107988	Lead_CeresClone107988
SEQ ID NO: 2017	109912	Identifier: Ceres CLONE ID no. 109912	Lead_CeresClone109912
SEQ ID NO: 2020	154718	Identifier: Ceres CLONE ID no. 154718	Lead_CeresClone154718
SEQ ID NO: 2024	226122	Identifier: Ceres CLONE ID no. 226122	Lead_CeresClone226122
SEQ ID NO: 2039	691319	Identifier: Ceres CLONE ID no. 691319	Lead_CeresClone691319
SEQ ID NO: 2045	641	Identifier: Ceres CLONE ID no. 641	Lead_CeresClone641
SEQ ID NO: 2050	3819	Identifier: Ceres CLONE ID no. 3819	Lead_CeresClone3819
SEQ ID NO: 2069	3853	Identifier: Ceres CLONE ID no. 3853	Lead_CeresClone3853
SEQ ID NO: 2074	8133	Identifier: Ceres CLONE ID no. 8133	Lead_CeresClone8133
SEQ ID NO: 2079	15343	Identifier: Ceres CLONE ID no. 15343	Lead_CeresClone15343
SEQ ID NO: 2084	22007	Identifier: Ceres CLONE ID no. 22007	Lead_CeresClone22007
SEQ ID NO: 2088	23771	Identifier: Ceres CLONE ID no. 23771	Lead_CeresClone23771
SEQ ID NO: 2101	27197	Identifier: Ceres CLONE ID no. 27197	Lead_CeresClone27197
SEQ ID NO: 2111	33802	Identifier: Ceres CLONE ID no. 33802	Lead_CeresClone33802
SEQ ID NO: 2117	34210	Identifier: Ceres CLONE ID no. 34210	Lead_CeresClone34210
SEQ ID NO: 2130	38757	Identifier: Ceres CLONE ID no. 38757	Lead_CeresClone38757
SEQ ID NO: 2135	38785	Identifier: Ceres CLONE ID no. 38785	Lead_CeresClone38785
SEQ ID NO: 2141	38843	Identifier: Ceres CLONE ID no. 38843	Lead_CeresClone38843
SEQ ID NO: 2148	39127	Identifier: Ceres CLONE ID no. 39127	Lead_CeresClone39127
SEQ ID NO: 2154	95855	Identifier: Ceres CLONE ID no. 95855	Lead_CeresClone95855
SEQ ID NO: 2158	99763	Identifier: Ceres CLONE ID no. 99763	Lead_CeresClone99763
SEQ ID NO: 2170	267657	Identifier: Ceres CLONE ID no. 267657	Lead_CeresClone267657
SEQ ID NO: 2173	545208	Identifier: Ceres CLONE ID no. 545208	Lead_CeresClone545208
SEQ ID NO: 2178	546490	Identifier: Ceres CLONE ID no. 546490	Lead_CeresClone546490
SEQ ID NO: 2181	566317	Identifier: Ceres CLONE ID no. 566317	Lead_CeresClone566317
SEQ ID NO: 2188	28003	Identifier: Ceres CLONE ID no. 28003	Lead_CeresClone28003
SEQ ID NO: 2192	225200	Identifier: Ceres CLONE ID no. 225200	Lead_CeresClone225200
SEQ ID NO: 2200	301326	Identifier: Ceres CLONE ID no. 301326	Lead_CeresClone301326
SEQ ID NO: 2203	681088	Identifier: Ceres CLONE ID no. 681088	Lead_CeresClone681088
SEQ ID NO: 2210	681222	Identifier: Ceres CLONE ID no. 681222	Lead_CeresClone681222
SEQ ID NO: 2219	11975	Identifier: Ceres CLONE ID no. 11975	Lead_CeresClone11975
SEQ ID NO: 2233	14105	Identifier: Ceres CLONE ID no. 14105	Lead_CeresClone14105
SEQ ID NO: 2240	156807	Identifier: Ceres CLONE ID no. 156807	Lead_CeresClone156807
SEQ ID NO: 2245	228787	Identifier: Ceres CLONE ID no. 228787	Lead_CeresClone228787
SEQ ID NO: 2258	537272	Identifier: Ceres CLONE ID no. 537272	Lead_CeresClone537272
SEQ ID NO: 2275	608818	Identifier: Ceres CLONE ID no. 608818	Lead_CeresClone608818
SEQ ID NO: 2283	5055	Identifier: Ceres CLONE ID no. 5055	Lead_CeresClone5055
SEQ ID NO: 2293	331626	Identifier: Ceres CLONE ID no. 331626	Lead_CeresClone331626
SEQ ID NO: 2307	35742	Identifier: Ceres CLONE ID no. 35742	Lead_CeresClone35742
SEQ ID NO: 2312	22382	Identifier: Ceres CLONE ID no. 22382	Lead_CeresClone22382
SEQ ID NO: 2328	36334	Identifier: Ceres CLONE ID no. 36334	Lead_CeresClone36334
SEQ ID NO: 2334	102248	Identifier: Ceres CLONE ID no. 102248	Lead_CeresClone102248
SEQ ID NO: 2347	157709	Identifier: Ceres CLONE ID no. 157709	Lead_CeresClone157709
SEQ ID NO: 2350	24885	Identifier: Ceres CLONE ID no. 24885	Lead_CeresClone24885
SEQ ID NO: 2356	27810	Identifier: Ceres CLONE ID no. 27810	Lead_CeresClone27810
SEQ ID NO: 2370	40708	Identifier: Ceres CLONE ID no. 40708	Lead_CeresClone40708
SEQ ID NO: 2381	116117	Identifier: Ceres CLONE ID no. 116117	Lead_CeresClone116117
SEQ ID NO: 2386	538933	Identifier: Ceres CLONE ID no. 538933	Lead_CeresClone538933
SEQ ID NO: 2397	519	Identifier: Ceres CLONE ID no. 519	Lead_CeresClone519
SEQ ID NO: 2414	11843	Identifier: Ceres CLONE ID no. 11843	Lead_CeresClone11843
SEQ ID NO: 2423	14033	Identifier: Ceres CLONE ID no. 14033	Lead_CeresClone14033
SEQ ID NO: 2428	29009	Identifier: Ceres CLONE ID no. 29009	Lead_CeresClone29009
SEQ ID NO: 2431	32574	Identifier: Ceres CLONE ID no. 32574	Lead_CeresClone32574
SEQ ID NO: 2437	32612	Identifier: Ceres CLONE ID no. 32612	Lead_CeresClone32612
SEQ ID NO: 2453	36094	Identifier: Ceres CLONE ID no. 36094	Lead_CeresClone36094
SEQ ID NO: 2460	115966	Identifier: Ceres CLONE ID no. 115966	Lead_CeresClone115966
SEQ ID NO: 2472	121021	Identifier: Ceres CLONE ID no. 121021	Lead_CeresClone121021
SEQ ID NO: 2476	248859	Identifier: Ceres CLONE ID no. 248859	Lead_CeresClone248859
SEQ ID NO: 2481	266142	Identifier: Ceres CLONE ID no. 266142	Lead_CeresClone266142
SEQ ID NO: 2492	609573	Identifier: Ceres CLONE ID no. 609573	Lead_CeresClone609573
SEQ ID NO: 2503	3968	Identifier: Ceres CLONE ID no. 3968	Lead_CeresClone3968
SEQ ID NO: 2509	98850	Identifier: Ceres CLONE ID no. 98850	Lead_CeresClone98850

Claims

1. An isolated nucleic acid molecule comprising:

a) a nucleic acid having a nucleotide sequence which encodes an amino acid sequence exhibiting at least 85% sequence identity to an amino acid sequence in the Sequence Listing or in the ortholog alignments of FIG. 1;

b) a nucleic acid which is a complement of a nucleotide sequence according to paragraph (a);

c) a nucleic acid which is the reverse of the nucleotide sequence according to subparagraph (a), such that the reverse nucleotide sequence has a sequence order which is the reverse of the sequence order of the nucleotide sequence according to subparagraph (a); or

d) a nucleic acid capable of hybridizing to a nucleic acid according to any one of paragraphs (a)-(c), under conditions that permit formation of a nucleic acid duplex at a temperature from about 40° C. and 48° C. below the melting temperature of the nucleic acid duplex.

2. The isolated nucleic acid molecule according to claim 1, which has the nucleotide sequence according to any polynucleotide sequence in the Sequence Listing.

3. The isolated nucleic acid molecule according to claim 1, wherein said amino acid sequence comprises any polypeptide sequence in the Sequence Listing or in the ortholog alignments of FIG. 1.

4. A vector construct comprising:

a) a first nucleic acid having a regulatory sequence capable of causing transcription and/or translation in a plant; and

b) a second nucleic acid having the sequence of the isolated nucleic acid molecule according to claim 1;

wherein said first and second nucleic acids are operably linked and

wherein said second nucleic acid is heterologous to any element in said vector Construct.

5. The vector construct according to claim 4, wherein said first nucleic acid is native to said second nucleic acid.

6. The vector construct according to claim 4, wherein said first nucleic acid is heterologous to said second nucleic acid.

7. A host cell comprising an isolated nucleic acid molecule according to claim 1 wherein said nucleic acid molecule is flanked by exogenous sequence.

8. A host cell comprising a vector construct according to claim 4.

9. An isolated polypeptide comprising an amino acid sequence exhibiting at least 85% sequence identity of an amino acid sequence of the Sequence Listing or in the ortholog alignments of FIG. 1.

10. A method of introducing an isolated nucleic acid into a host cell comprising:

a) providing an isolated nucleic acid molecule according to claim 1; and

b) contacting said isolated nucleic with said host cell under conditions that permit insertion of said nucleic acid into said host cell.

11. A method of transforming a host cell which comprises contacting a host cell with a vector construct according to claim 4.

12. A method for detecting a nucleic acid in a sample which comprises:

a) providing an isolated nucleic acid molecule according to claim 1;

b) contacting said isolated nucleic acid molecule with a sample under conditions which permit a comparison of the sequence of said isolated nucleic acid molecule with the sequence of DNA in said sample; and

c) analyzing the result of said comparison.

13. A host cell or organism which comprises a nucleic acid molecule according to claim 1 which is exogenous or heterologous to said plant or plant cell.

14. A host cell or organism according to claim 13, which is a plant, plant cell, plant material or seed of a plant.

15. A plant which has been regenerated from a plant cell or seed according to claim 14.

16. A plant, plant cell, plant material or seed of a plant which comprises a nucleic acid molecule according to claim 1, wherein said plant has improved characteristics as compared to a wild-type plant cultivated under the same conditions.

17. The plant, plant cell, plant material or seed of a plant according to claim 16, wherein the improved characteristic is the one associated with and described in the miscellaneous feature section of the sequence listing for the particular sequence.

18. A method for improving plant characteristics in a plant comprising transforming a plant with a nucleic acid sequence according to claim 1.

19. A transgenic plant having a gene construct comprising a nucleic acid according to claim 1 encoding a component operably linked to a plant promoter so that the component is ectopically overexpressed in the transgenic plant, and the transgenic plant exhibits:

i) faster rate of growth,

ii) greater fresh or dry weight at maturation,

iii) greater fruit or seed yield,

iv) higher tolerance to pH,

v) higher tolerance to low phosphate concentration, or

vi) higher tolerance to low nitrogen concentration

than a progenitor plant which does not contain the polynucleotide construct, when the transgenic plant and the progenitor plant are cultivated under identical environmental conditions, wherein the component is any one of the polypeptides set forth in the Sequence Listing or in the ortholog alignment of FIG. 1.