Patent Application
20110277183
This invention relates to the field of plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for altering the plant architecture characteristics.
Crop plants with desirable architecture are able to produce increased yields (Yonghong Wang, Jiayang Li. (2008) Molecular Basis of Plant Architecture. Annu. Rev. Plant Biol. 59, 253-279). Plant height, an important component of plant architecture, not only contributes to crop yields, but also highly correlates with biomass yield. Furthermore, the increasing demand for lignocellulosic biomass for the production of biofuels may lead to a shift in desirable plant architecture characteristics (Maria G. Salas Fernandez, Philip W. Becraft, Yanhai Yin, Thomas Lubberstedt. (2009) From Dwarves to Giants? Plant Height Manipulation for Biomass Yield. Trends in Plant Science. 14, 454-461). Shorter plants can be better against lodging, while more erect leaves or smaller leaf angle can lead to high planting density adaptation and yield enhancement. Taller plants can be beneficial for increased demand for lignocellulosic biomass production.
Most phenotypic variation occurring in natural plant populations is continuous and is affected by multiple genes. Very few genes have been known that alter plant architecture characteristics at a single gene level.
The availability of such single genes would greatly decrease the complexity of developing crops with enhanced plant architecture characteristics. Thus, it is desirable to provide compositions and methods useful in altering plant architecture characteristics.
The present invention includes:
In one embodiment, a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, and wherein said plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising said recombinant DNA construct.
In another embodiment, a plant comprising in its genome a recombinant DNA construct comprising a suppression DNA construct comprising at least one regulatory element operably linked to: (i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52 or (B) a full complement of the nucleic acid sequence of (i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Squatty-Crinkle-Leaf polypeptide; and wherein said plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising said recombinant DNA construct.
In another embodiment, any of the plants of the present invention wherein said at least one plant architecture characteristic is selected from the group consisting of plant height, stalk length, internode length, leaf angle, leaf length, leaf surface, leaf width, leaf hair number, leaf hair volume, leaf initiation rate, leaf morphology, seedling size, and seedling growth rate.
In another embodiment, any of the plants of the present invention wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane, and switchgrass.
In another embodiment, seed of any of the plants of the present invention, wherein said seed comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, and wherein a plant produced from said seed exhibits an alteration in at least one plant architecture characteristic selected from the group consisting of: plant height, stalk length, internode length, leaf angle, leaf length, leaf surface, leaf width, leaf hair number, leaf hair volume, leaf initiation rate, leaf morphology, seedling size, and seedling growth rate, when compared to a control plant not comprising said recombinant DNA construct. The alteration in at least one plant architecture characteristic can be either an increase or a decrease in a plant architecture characteristic.
In another embodiment, a method of altering at least one plant architecture characteristic in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) obtaining a progeny plant derived from the transgenic plant of step (b), wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits an alteration in at least one plant architecture characteristic when compared to a control plant not comprising the recombinant DNA construct.
In another embodiment, a method of altering at least one plant architecture characteristic in a plant, comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: (i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52 or (B) a full complement of the nucleic acid sequence of (i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Squatty-Crinkle-Leaf polypeptide; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and (c) determining whether the transgenic plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising the suppression DNA construct. Optionally, said method further comprises: (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (e) determining whether the progeny plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising the suppression DNA construct.
In another embodiment, a method of determining an alteration of at least one plant architecture characteristics in a plant, comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52; (b) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) determining whether the progeny plant exhibits an alteration of at least one plant architecture characteristics when compared to a control plant not comprising the recombinant DNA construct.
In another embodiment, a method of selecting a maize plant or germplasm that displays an alteration of at least one plant architecture characteristic comprising: a) obtaining DNA accessible for analysis; b) detecting the presence or absence of at least one allele of a marker locus comprising a point mutation at position 20 or 206 of SEQ ID NO: 53; and, c) selecting said maize plant or germplasm that comprises a point mutation at position 20 or 206 of SEQ ID NO: 53.
In another embodiment, a method of selecting a maize plant or germplasm that displays an alteration of at least one plant architecture characteristic comprising: a) obtaining DNA accessible for analysis; b) detecting the presence or absence of at least one allele of a marker locus comprising a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, c) selecting said maize plant or germplasm that comprises a point mutation at position 20 or 206 of SEQ ID NO: 53 and wherein the at least one allele of the marker locus is located on a DNA interval between BAC c0137A18, or a nucleotide sequence that is 95% identical to BAC c0137A18, and BAC c0427D16, or a nucleotide sequence that is 95% identical to BAC c0427D16, based on the Clustal V method of alignment. Optionally, the at least one allele of the marker locus is on or within SEQ ID NO:39 or 52.
In another embodiment, a method of selecting a maize plant or germplasm that displays an altered plant architecture comprising: a) obtaining DNA accessible for analysis; b) detecting the presence of at least one allele of a first marker locus that is linked to and associated with an allele of a second marker locus, wherein the allele of the second marker locus comprises a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, c) selecting said maize plant or germplasm that comprises a point mutation at position 20 or 206, or both, of SEQ ID NO: 53.
In another embodiment, a method of marker assisted selection comprising: a) selecting a first maize plant that displays an alteration in at least one plant architecture characteristic comprising: i) obtaining DNA accessible for analysis; ii) detecting the presence of at least one allele of a first marker locus that is linked to and associated with an allele of a second marker locus, wherein the allele of the second marker locus comprises a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, iii) selecting said first maize plant that comprises a point mutation at position 20 or 206, or both, of SEQ ID NO: 53; b) crossing said first maize plant to a second maize plant; c) evaluating the progeny for at least said one allele of a first marker locus; and d) selecting progeny plants that possess at least said one allele of a first marker locus.
In another embodiment, any of the methods of the present invention wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane, and switchgrass. In another embodiment, an isolated polynucleotide comprising: a nucleotide sequence encoding a polypeptide with plant architecture altering activity wherein, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, the polypeptide has an amino acid sequence of at least 99% sequence identity when compared to SEQ ID NO:52; or (b) a full complement of the nucleotide sequence, wherein the full complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. The polypeptide may comprise the amino acid sequence of SEQ ID NO:52. The nucleotide sequence may comprise the nucleotide sequence of SEQ ID NO:51.
In another embodiment, a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence, or a cell, a plant, or a seed comprising the recombinant DNA construct. The cell may be eukaryotic, e.g., a yeast, insect, or plant cell, or prokaryotic, e.g., a bacterial cell.
The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.
The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (2):345-373 (1984), which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.
Table 1 lists the sequences described herein that are associated with the PHM markers, along with the corresponding identifiers (SEQ ID NO:XX) as used in the attached Sequence Listing.
SEQ ID NO:21 is the nucleotide sequence of primer c0137A18-B1_F.
SEQ ID NO:22 is the nucleotide sequence of primer c0137A18-B1_R.
SEQ ID NO:23 is the nucleotide sequence of primer c0427D16-D1_F.
SEQ ID NO:24 is the nucleotide sequence of primer c0427D16-D1_R.
SEQ ID NO:25 is the nucleotide sequence of primer c0427D16-A1_F.
SEQ ID NO:26 is the nucleotide sequence of primer c0427D16-A1_R.
SEQ ID NO:27 is the nucleotide sequence of primer PHM589962-3_F.
SEQ ID NO:28 is the nucleotide sequence of primer PHM589962-3_R.
SEQ ID NO:29 is the nucleotide sequence of primer PHM589962-4_F.
SEQ ID NO:30 is the nucleotide sequence of primer PHM589962-4_R.
SEQ ID NO:31 is the genomic nucleotide sequence of wild type maize (Zea mays) Squatty-Crinkle-Leaf (SCL) gene.
SEQ ID NO:32 is the genomic nucleotide sequence of the mutant Squatty-Crinkle-Leaf (SCL) gene from maize SCL-338 mutant.
SEQ ID NO:33 is the genomic nucleotide sequence of the mutant Squatty-Crinkle-Leaf (SCL) gene from maize SCL-474 mutant.
SEQ ID NO:34 is the nucleotide sequence of primer CDS1-F.
SEQ ID NO:35 is the nucleotide sequence of primer CDS1-R.
SEQ ID NO:36 is the nucleotide sequence (coding region) of the wild type maize encoding Squatty-Crinkle-Leaf (SCL) polypeptide.
SEQ ID NO:37 is the nucleotide sequence (coding region) of the dominant splicing variant of maize SCL-338 mutant encoding a Squatty-Crinkle-Leaf (SCL) polypeptide.
SEQ ID NO:38 is the nucleotide sequence (coding region) of the dominant splicing variant of maize SCL-474 mutant encoding a Squatty-Crinkle-Leaf (SCL) polypeptide.
SEQ ID NO:39 is the amino acid sequence of the wild type maize encoding a Squatty-Crinkle-Leaf (SCL) polypeptide.
SEQ ID NO:40 corresponds to NCBI GI No. 164421987, which is the amino acid sequence of AP2/EREBP-like protein from Otyza sativa Indica Group.
SEQ ID NO:41 corresponds to NCBI GI No. 54287602, which is the amino acid sequence of a putative AP2 domain transcription factor from Otyza sativa Japonica.
SEQ ID NO:42 corresponds to NCBI GI No. 21593696, which is the amino acid sequence of a putative AP2 domain transcription factor from Arabidopsis thaliana.
SEQ ID NO:43 corresponds to NCBI GI No. 18405784, which is the amino acid sequence of a putative protein from Arabidopsis thaliana.
SEQ ID NO:44 corresponds to NCBI GI No. 224138066, which is the amino acid sequence of an AP2 domain-containing transcription factor from Populus trichocarpa.
SEQ ID NO:45 corresponds to NCBI GI No. 224090105, which is the amino acid sequence of an AP2 domain-containing transcription factor from Populus trichocarpa.
SEQ ID NO:46 is the nucleotide sequence of PHP23236, a destination vector for use with Gaspe Flint derived maize lines.
SEQ ID NO:47 is the nucleotide sequence of PHP10523 (Komari et al., Plant J. 10:165-174 (1996); NCBI General Identifier No. 59797027).
SEQ ID NO: 48 is the nucleotide sequence of PHP29634, destination vector for use with Gaspe Flint derived maize lines.
SEQ ID NO: 49 is amino acid sequence encoded by the dominant splicing variant of the Squatty-Crinkle-Leaf (SCL) of maize SCL-338 mutant.
SEQ ID NO:50 is the amino acid sequence encoded by the dominant splicing variant of the Squatty-Crinkle-Leaf (SCL) of maize SCL-474 mutant.
SEQ ID NO:51 is a nucleotide sequence (coding region) of a wild type maize encoding a Squatty-Crinkle-Leaf (SCL) polypeptide present in clone p0031.ccmau15r-fis. This nucleotide sequence constitutes a variant of SEQ ID NO:36.
SEQ ID NO:52 is the amino acid sequence of the wild type SCL polypeptide encoded by SEQ ID NO:51.
SEQ ID NO:53 is the nucleotide sequence of a 250 bp fragment of the wild type maize (Zea mays) Squatty-Crinkle-Leaf (SCL) gene comprising the loci corresponding to the point mutation at position 1919 and 2105 of SEQ ID NO:31. Position 1919 of SEQ ID NO:31 corresponds to position 20 of SEQ ID NO:53 while position 2105 of SEQ ID NO:31 corresponds to position 206 of SEQ ID NO:53.
SEQ ID NO: 54 is the nucleotide sequence of the SCL MPSS tag.
The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants; reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.
Additionally, as used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Thus, for example, a nucleic acid comprising a particular sequence many possess nucleotides beyond those specifically recited. Additionally, the term “comprising” is intended to include examples encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.”
The following definitions are provided as an aid to understand this invention.
As used herein:
“Arabidopsis” and “Arabidopsis thaliana” are used interchangeably herein, unless otherwise indicated.
An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.
The term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.
An “amplicon” is an amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).
The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.
The term “assemble” applies to BACs and their propensities for coming together to form contiguous stretches of DNA. A BAC “assembles” to a contig based on sequence alignment, if the BAC is sequenced, or via the alignment of its BAC fingerprint to the fingerprints of other BACs. The assemblies can be found using the Maize Genome Browser, which is publicly available on the internet.
An allele is “associated with” a trait when it is linked to it and when the presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.
A “BAC”, or bacterial artificial chromosome, is a cloning vector derived from the naturally occurring F factor of Escherichia coli. BACs can accept large inserts of DNA sequence. In maize, a number of BACs, each containing a large insert of maize genomic DNA, have been assembled into contigs (overlapping contiguous genetic fragments, or “contiguous DNA”).
“Backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents.
A centimorgan (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.
The term “complement” refers to a nucleotide sequence that is complementary to a given nucleotide sequence, i.e., the sequences are related by the base-pairing rules.
A “chromosome” can also be referred to as a “linkage group.”
The term “contiguous DNA” refers to overlapping contiguous genetic fragments.
The term “crossed” or “cross” means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant). The term “crossing” refers to the act of fusing gametes via pollination to produce progeny.
An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from a cDNA library and therefore is a sequence which has been transcribed. An EST is typically obtained by a single sequencing pass of a cDNA insert. The sequence of an entire cDNA insert is termed the “Full-Insert Sequence” (“FIS”). A “Contig” sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS, and PCR sequence. A sequence encoding an entire or functional protein is termed a “Complete Gene Sequence” (“CGS”) and can be derived from an FIS or a contig.
A “favorable allele” is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, e.g., an alteration of at least one plant architecture characteristic, and that allows the identification of plants that have the agronomically desirable phenotype. A “favorable” allele of a marker is a marker allele that segregates with the favorable phenotype.
A favorable allelic form of a chromosome segment is a chromosome segment that includes a nucleotide sequence that contributes to superior agronomic performance at one or more genetic loci physically located on the chromosome segment. “Allele frequency” refers to the frequency (proportion or percentage) of an allele within a population, or a population of lines. One can estimate the allele frequency within a population by averaging the allele frequencies of a sample of individuals from that population.
An allele “positively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele. An allele negatively correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.
A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them, and recombinations between loci can be detected using a variety of markers. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and the genetic distances between markers can differ from one genetic map to another. For example, 10 cM on the internally derived genetic map (also referred to herein as “PHB” for Pioneer Hi-Bred) is roughly equivalent to 25-30 cM on the IBM2 2005 neighbors frame map (a high resolution map available on maize GDB). However, information can be correlated from one map to another using a general framework of common markers. One of ordinary skill in the art can use the framework of common markers to identify the positions of markers and loci of interest on each individual genetic map. A comparison of marker positions between the internally derived genetic map and the IBM2 neighbors genetic map, for example, can be seen in Table 6.
The term “Genetic Marker” shall refer to any type of nucleic acid based marker, including but not limited to, Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeat (SSR), Random Amplified Polymorphic DNA (RAPD), Cleaved Amplified Polymorphic Sequences (CAPS) (Rafalski and Tingey, 1993, Trends in Genetics 9:275-280), Amplified Fragment Length Polymorphism (AFLP) (Vos et al., 1995, Nucleic Acids Res. 23:4407-4414), Single Nucleotide Polymorphism (SNP) (Brookes, 1999, Gene 234:177-186), Sequence Characterized Amplified Region (SCAR) (Paran and Michelmore, 1993, Theor. Appl. Genet. 85:985-993), Sequence Tagged Site (STS) (Onozaki et al., 2004, Euphytica 138:255-262), Single Stranded Conformation Polymorphism (SSCP) (Orita et al., 1989, Proc Natl Acad Sci USA 86:2766-2770), Inter-Simple Sequence Repeat (ISSR) (Blair et al., 1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon Amplified Polymorphism (IRAP), Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) (Kalendar et al., 1999, Theor. Appl. Genet. 98:704-711), an RNA cleavage product (such as a Lynx tag), and the like.
“Genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits following meiosis.
The term “genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome.
“Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed, or tissues from which new plants may be grown, or plant parts, such as leafs, stems, pollen, or cells that can be cultured into a whole plant.
A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to a series of polymorphisms with a specific sequence, such as a marker locus, or a series of polymorphisms across multiple sequences, e.g., multiple marker loci.
A “heterotic group” comprises a set of genotypes that perform well when crossed with genotypes from a different heterotic group (Hallauer et al., (1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley (ed.) Corn and corn improvement). Inbred lines are classified into heterotic groups, and are further subdivided into families within a heterotic group, based on several criteria such as pedigree, molecular marker-based associations, and performance in hybrid combinations (Smith et al., (1990) Theor. Appl. Gen. 80:833-840). The two most widely used heterotic groups in the United States are referred to as “Iowa Stiff Stalk Synthetic” (BSSS) and “Lancaster” or “Lancaster Sure Crop” (sometimes referred to as NSS, or non-Stiff Stalk).
The term “heterozygous” means a genetic condition wherein different alleles reside at corresponding loci on homologous chromosomes.
The term “homozygous” means a genetic condition wherein identical alleles reside at corresponding loci on homologous chromosomes.
The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.
“Hybridization” or “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.
The term “hybridize” means to form base pairs between complementary regions of nucleic acid strands.
An “IBM genetic map” refers to any of following maps: IBM, IBM2, IBM2 neighbors, IBM2 FPCO507, IBM2 2004 neighbors, IBM2 2005 neighbors, or IBM2 2005 neighbors frame. IBM genetic maps are based on a B73×Mo17 population in which the progeny from the initial cross were random-mated for multiple generations prior to constructing recombinant inbred lines for mapping. Newer versions reflect the addition of genetic and BAC mapped loci as well as enhanced map refinement due to the incorporation of information obtained from other genetic maps.
The term “inbred” refers to a line that has been bred for genetic homogeneity.
The term “indel” refers to an insertion or deletion, wherein one line may be referred to as having an insertion relative to a second line, or the second line may be referred to as having a deletion relative to the first line.
The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a selected allele of a marker, a QTL, a transgene, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.
The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times. In introgressing or backcrossing, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed.
The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al., (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross gives rise to the F1 generation; the term “BC1” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.
As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus (for example, a locus for an alteration of at least one plant architecture characteristic). The linkage relationship between a molecular marker and a phenotype (for example, an alteration of at least one plant architecture characteristic) is given as a “probability” or “adjusted probability.” Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units (or cM). In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, “closely linked loci” such as a marker locus and a second locus display an inter-locus recombination frequency of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. Two closely linked markers on the same chromosome can be positioned 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, or 0.25 cM or less from each other.
The term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency (in the case of co-segregating traits, the loci that underlie the traits are in sufficient proximity to each other). Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group.) As used herein, linkage can be between two markers, or alternatively between a marker and a phenotype. A marker locus can be “associated with” (linked to) a trait, e.g., an alteration of at least one plant architecture characteristic. The degree of linkage of a molecular marker to a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that molecular marker with the phenotype.
Linkage disequilibrium is most commonly assessed using the measure r2, which is calculated using the formula described by Hill, W. G. and Robertson, A, Theor. Appl. Genet. 38:226-231 (1968). When r2=1, complete LD exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency. Values for r2 above ⅓ indicate sufficiently strong LD to be useful for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkage disequilibrium when r2 values between pairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.
As used herein, “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).
A “locus” is a position on a chromosome where a gene or marker is located.
The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science 255:803-804 (1992)) is used in interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage.
“Maize” refers to a plant of the Zea mays L. ssp. mays and is also known as corn.
The term “maize plant” includes: whole maize plants, maize plant cells, maize plant protoplast, maize plant cell or maize tissue cultures from which maize plants can be regenerated, maize plant calli, and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips, and the like.
A “marker” is a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference. A marker can be derived from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence.
Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well established in the art. These include, e.g., DNA sequencing, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).
A “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus. Alternatively, marker alleles designated with a number, represent the specific combination of alleles, also referred to as a “marker haplotype”, at that specific marker locus.
“Marker assisted selection” (“MAS”) is a process by which individual plants are selected based on marker genotypes.
“Marker assisted counter-selection” is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.
A “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL, that are genetically or physically linked to the marker locus.
A “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus (“all or a portion” of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are “complementary” when they specifically “hybridize”, or pair, in solution, e.g., according to Watson-Crick base pairing rules.
The term “molecular marker” may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are “complementary” when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the non-collinear region described herein. This is because the insertion region is, by definition, a polymorphism vis a vis a plant without the insertion. Thus, the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g., SNP technology is used in the examples provided herein.
The terms “phenotype,” or “phenotypic trait,” or “trait” refer to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. The phenotype, phenotypic trait, or trait can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait.” In other cases, a phenotype is the result of several genes.
A “physical map” of the genome is a map showing the linear order of identifiable landmarks (including genes, markers, etc.) on chromosome DNA. However, in contrast to genetic maps, the distances between landmarks are absolute (for example, measured in base pairs or isolated and overlapping contiguous genetic fragments) and not based on genetic recombination.
A “plant” can be a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same. A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
A “polymorphism” is a variation in the DNA that is too common to be due merely to new mutation. A polymorphism must have a frequency of at least 1% in a population. A polymorphism can be a single nucleotide polymorphism, or SNP, or an insertion/deletion polymorphism, also referred to herein as an “indel”.
The “probability value” or “p-value” is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker allele is random. Thus, the lower the probability score, the greater the likelihood that a phenotype and a particular marker will co-segregate. In some aspects, the probability score is considered “significant” or “nonsignificant”. In some embodiments, a probability score of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of co-segregation. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05, less than 0.01, or less than 0.001.
Each “PHM” marker represents two sets of primers (external and internal) that, when used in a nested PCR, amplify a specific piece of DNA. The external set is used in the first round of PCR, after which the internal sequences are used for a second round of PCR on the products of the first round. This increases the specificity of the reaction.
SNP markers can also be developed for specific polymorphisms identified using the PHM markers and the nested PCR analysis. These SNP markers can be specifically designed for use with the Invader® (Third Wave Technologies) platform.
A “production marker” or “production SNP marker” is a marker that has been developed for high-throughput purposes. Production SNP markers are developed for specific polymorphisms identified using PHM markers and the nested PCR analysis.
The term “progeny” refers to the offspring generated from a cross.
A “progeny plant” is generated from a cross between two plants.
The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a phenotypic trait in at least one genetic background, e.g., in at least one breeding population. QTLs are closely linked to the gene or genes that underlie the trait in question.
A “topeross test” is a progeny test derived by crossing each parent with the same tester, usually a homozygous line. The parent being tested can be an open-pollinated variety, a cross, or an inbred line.
The phrase “under stringent conditions” refers to conditions under which a probe or polynucleotide will hybridize to a specific nucleic acid sequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.
An “unfavorable allele” of a marker is a marker allele that segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants that can be removed from a breeding program or planting.
“SCL” and “Squatty-Crinkle-Leaf” are used interchangeably herein. The term “Squatty” refers to short and thicker in stature. The term “Crinkle” refers to the wrinkled leaf surface of the leaf.
“Plant architecture characteristic” refers to a measurable parameter including, but not limited to, plant height, stalk length, internode length, leaf angle, leaf length, leaf surface, leaf width, leaf hair number, leaf hair volume, leaf initiation rate, leaf morphology, seedling size, and seedling growth rate.
An “alteration in at least one plant architecture characteristic” of a plant is measured relative to a reference or control plant. Plant architecture characteristics include, for example, plant height, stalk length, internode length, leaf angle, leaf length, leaf surface, leaf width, leaf hair number, leaf hair volume, leaf initiation rate, leaf morphology, seedling size, and seedling growth rate. Typically, when a transgenic plant comprising a recombinant DNA construct or suppression DNA construct in its genome exhibits an alteration in at least one plant architecture characteristic relative to a reference or control plant, the reference or control plant does not comprise in its genome the recombinant DNA construct or suppression DNA construct.
Increased leaf surface may be of particular interest. Increasing leaf surface can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in total plant photosynthesis is typically achieved by increasing leaf area of the plant. Additional photosynthetic capacity may be used to increase the yield derived from particular plant tissue, including the leaves, roots, fruits, or seed, or permit the growth of a plant under decreased light intensity or under high light intensity.
Increasing plant height may be beneficial to crops and ornamental plants, where the ability to provide taller varieties would be highly desirable. For many plants, including fruit-bearing trees, trees that are used for lumber production, or trees and shrubs that serve as view or wind screens, increased stature provides improved benefits in the forms of greater yield or improved screening. Taller plants are also desirable for increased lignocellulosic biomass production for the production of biofuels.
Decreased plant height may be desirable to reduce lodging in crops.
Decreased leaf angle may be beneficial to crops and plants to allow for good light penetration in the canopy while allowing for increased plant density and yield when compared to control plants with greater leaf angle.
“Transgenic” refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
“Genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.
“Progeny” comprises any subsequent generation of a plant.
“Transgenic plant” includes reference to a plant that comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
“Heterologous” with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
“Polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
“Polypeptide,” “peptide,” “amino acid sequence,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.
“Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell.
“cDNA” refers to a DNA that is complementary to and synthesized from an mRNA template using, e.g., the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using, e.g., the Klenow fragment of DNA polymerase I.
“Mature” protein refers to a post-translationally processed polypeptide, i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed.
“Precursor” protein refers to the primary product of translation of mRNA, i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.
“Isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free of or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
“Recombinant” refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. “Recombinant” also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
“Recombinant DNA construct” refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
The terms “entry clone” and “entry vector” are used interchangeably herein.
“Regulatory sequences” 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 may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
“Promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.
“Promoter functional in a plant” is a promoter capable of controlling transcription in plant cells whether or not its origin is from a plant cell.
“Tissue-specific promoter” and “tissue-preferred promoter” are used interchangeably, and refer to a promoter that is expressed predominantly, but not necessarily exclusively, in one tissue or organ, but that may also be expressed in one specific cell.
“Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events.
“Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
“Expression” refers to the production of a functional product. For example, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
“Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
A “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
“Transformation” as used herein refers to both stable transformation and transient transformation.
“Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
“Transient transformation” refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
“Allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant, that plant is hemizygous at that locus.
The “Clustal V method of alignment” corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., (1992) Comput. Appl. Biosci. 8:189-191) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.
Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).
Molecular markers can be used in a variety of plant breeding applications (e.g., see Staub et al., (1996) Hortscience 31: 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS). A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay, e.g., many disease resistance traits, or occurs at a late stage in plant development, e.g., kernel characteristics. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a “perfect marker.”
When a gene is introgressed by MAS, it is not only the gene that is introduced, but also the flanking regions (Gepts. (2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. This “linkage drag” may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite maize line. This is also sometimes referred to as “yield drag.” The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al., (1998) Genetics 120:579-585). In classical breeding, it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al., (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will have experienced a crossover within 1 cM of the gene, based on a single meiosis map distance. Markers will allow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley at al., supra). When the exact location of a gene is known, a series of flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.
The availability of integrated linkage maps of the maize genome containing increasing densities of public maize markers has facilitated maize genetic mapping and MAS. See, e.g., the IBM2 Neighbors maps, which are available online on the MaizeGDB website.
The key components to the implementation of MAS are: (i) Defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and/or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.
SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al., (1994) Theoretical and Applied Genetics, 88:1-6). Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al., (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis; a practical guide. Academic press. Pp 75-135).
Various types of SSR markers can be generated, and SSR profiles from resistant lines can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment. An SSR service for maize is available to the public on a contractual basis by DNA Landmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.
Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al., (2002). Plant Mol Blot 48:539-547; Rafalski (2002b), supra).
SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide the highest genetic map resolution (Bhattramakki et al., (2002) Plant Mol Biol 8:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called “ultra-high-throughput” fashion, as they do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing, and coded spheres. Such methods have been reviewed, for example, in Gut (2001) Hum Mutat 17:475-492; Shi (2001) Clin Chem 47:164-172; Kwok (2000) Pharmacogenomics 1:95-100; Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs, including Masscode™ (Qiagen), Invader® (Third Wave Technologies), SnapShot® (Applied Biosystems), Taqman® (Applied Biosystems) and Beadarrays™ (Illumina).
A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al., (2002), BMC Genet. 3:19; Gupta et al., 2001; Rafalski (2002b); Plant Science 162:329-333). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene (see, for example, WO2003054229). Using automated high throughput marker detection platforms known to those of ordinary skill in the art makes this process highly efficient and effective.
Many of the primers listed herein can be used as FLP markers. These primers can also be used to convert these markers to SNP or other structurally similar or functionally equivalent markers (SSRs, CAPs, indels, etc), in the same regions. One very productive approach for SNP conversion is described by Rafalski (2002a) Current opinion in plant biology 5 (2): 94-100 and also Rafalski (2002b) Plant Science 162: 329-333. Using PCR, the primers are used to amplify DNA segments from individuals (preferably inbred) that represent the diversity in the population of interest. The PCR products are sequenced directly in one or both directions. The resulting sequences are aligned and polymorphisms are identified. The polymorphisms are not limited to single nucleotide polymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats). Specifically with respect to the fine map information described herein, one can readily use the information provided herein to obtain additional polymorphic SNPs (and other markers) within the region amplified by the primers listed in this disclosure. Markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.
In addition to SSRs, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including, but not limited to, expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid based markers.
Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).
Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and/or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the maize species, or even across other species that have been genetically or physically aligned with maize, such as rice, wheat, barley, or sorghum.
In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with an alteration of at least one plant architecture characteristic. Such markers are presumed to map near a gene or genes that give the plant an alteration of at least one plant architecture characteristic phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny.
The markers and intervals presented herein find use in MAS to select plants that demonstrate an alteration of at least one plant architecture characteristic.
Methods for selection can involve obtaining DNA accessible for analysis, detecting the presence (or absence) of either an identified marker allele or an unknown marker allele that is linked to and associated with an identified marker allele, and then selecting the maize plant or germplasm based on the allele detected.
Maize plant breeders desire combinations of desired genetic loci, such as those marker alleles associated with an alteration of at least one plant architecture characteristic, with genes for high yield and other desirable traits to develop improved maize varieties. Screening large numbers of samples by non-molecular methods (e.g., trait evaluation in maize plants) can be expensive, time consuming, and unreliable. Use of the polymorphic markers described herein, when genetically-linked to an alteration of at least one plant architecture characteristic, provide an effective method for selecting varieties with an alteration of at least one plant architecture characteristic in breeding programs. For example, one advantage of marker-assisted selection over field evaluations for alterations of plant architecture characteristics is that MAS can be done at any time of year, regardless of the growing season. Moreover, environmental effects are largely irrelevant to marker-assisted selection.
Another use of MAS in plant breeding is to assist the recovery of the recurrent parent genotype by backcross breeding. Backcross breeding is the process of crossing a progeny back to one of its parents or parent lines. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent (e.g., a parent having marker loci for an alteration of at least one plant architecture characteristic) into an otherwise desirable genetic background from the recurrent parent (e.g., an otherwise high yielding maize line). The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting introgressed variety. This is often necessary, because plants may be otherwise undesirable, e.g., due to low yield, low fecundity, or the like. In contrast, strains which are the result of intensive breeding programs may have excellent yield, fecundity or the like, merely being deficient in one desired trait such as an alteration of at least one plant architecture characteristic.
In marker assisted backcrossing of specific markers (and associated QTL) from a donor source, e.g., to an elite or exotic genetic background, one selects among backcross progeny for the donor trait and then uses repeated backcrossing to the elite or exotic line to reconstitute as much of the elite/exotic background's genome as possible.
Turning now to preferred embodiments:
Embodiments include isolated polynucleotides and polypeptides, recombinant DNA constructs useful for conferring the alteration of at least one plant architecture characteristic, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs.
As described herein, the SCL mutant plants are characterized by a small stature. Down regulating or silencing the wild type SCL gene in plants can result in smaller plants. The SCL mutants or transgenic plants with silenced SCL genes can also be used in a corn screening assay system, in which smaller plants are easier to handle and take less space to grow than larger plants. Also, from an agronomic value, increasing planting density has been a main approach to increase yield per acre in corn breeding. Shorter plants under higher planting density can be used to increase plant density as they are less prone to lodging. Furthermore, manipulating leaf angle to create more erect leaves is known to allow more light penetration to the lower canopy and thus enhance overall photosynthesis.
With regard to biomass production, it would be desirable to achieve a high plant stature and larger plants. As described herein this can be achieved by over-expressing the SCL gene. Over-expressing the gene can be used to increase plant or organ size, and increase yield.
Besides plant stature, modulating the level of SCL expression in plants by either down-regulation or over-expression may be used to alter specific organ size. For example, targeting the SCL gene to maize embryos may increase the embryo size and reduce tassel size.
The present invention includes the following isolated polynucleotides and polypeptides:
An isolated polynucleotide comprising: (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52; or (ii) a full complement of the nucleic acid sequence of (i), wherein the full complement and the nucleic acid sequence of (i) consist of the same number of nucleotides and are 100% complementary. Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs (including suppression DNA constructs) of the present invention. The polypeptide is preferably a Squatty-Crinkle-Leaf polypeptide. The polypeptide preferably has plant architecture altering activity.
An isolated polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52. The polypeptide is preferably a Squatty-Crinkle-Leaf polypeptide. The polypeptide preferably has plant architecture altering activity.
An isolated polynucleotide comprising (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:31, 36 or 51; or (ii) a full complement of the nucleic acid sequence of (i). Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs (including suppression DNA constructs) of the present invention. The polypeptide is preferably a Squatty-Crinkle-Leaf polypeptide. The polypeptide preferably has plant architecture altering activity.
In another embodiment, the present invention includes an isolated polynucleotide comprising: a nucleotide sequence encoding a polypeptide with plant architecture altering activity wherein, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, the polypeptide has an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% % sequence identity when compared to SEQ ID NO:52; or (b) a full complement of the nucleotide sequence, wherein the full complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. The polypeptide may comprise the amino acid sequence of SEQ ID NO:52. The nucleotide sequence may comprise the nucleotide sequence of SEQ ID NO:51.
In one aspect, the present invention includes recombinant DNA constructs (including suppression DNA constructs).
In one embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein the polynucleotide comprises (i) a nucleic acid sequence encoding an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:39 or 52; or (ii) a full complement of the nucleic acid sequence of (i).
In another embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide comprises (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:31, 36 or 51; or (ii) a full complement of the nucleic acid sequence of (i).
In another embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide encodes a Squatty-Crinkle-Leaf polypeptide. Preferably, the Squatty-Crinkle-Leaf polypeptide is from Zea mays, Glycine max, Glycine tabacina, Glycine sofa, Glycine tomentella, Arabidopsis thaliana, Oryza sativa, or Populus trichocarpa
In another aspect, the present invention includes suppression DNA constructs.
A suppression DNA construct preferably comprises at least one regulatory sequence (preferably a promoter functional in a plant) operably linked to (a) all or part of: (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, or (ii) a full complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Squatty-Crinkle-Leaf polypeptide; or (c) all or part of: (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:31, 36 or 51, or (ii) a full complement of the nucleic acid sequence of (c)(i). The suppression DNA construct preferably comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).
It is understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences. Alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes that result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
“Suppression DNA construct” is a recombinant DNA construct that, when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The terms “suppression,” “suppressing,” and “silencing”, used interchangeably herein, include lowering, reducing, declining, decreasing, inhibiting, eliminating or preventing. “Silencing” or “gene silencing” does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, and small RNA-based approaches.
A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.
Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence.
“Cosuppression” refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).
Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication No. WO 98/36083 published on Aug. 20, 1998).
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., Nature 391:806 (1998)). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., Trends Genet. 15:358 (1999)).
Small RNAs play an important role in controlling gene expression. Regulation of many developmental processes, including flowering, is controlled by small RNAs. It is now possible to engineer changes in gene expression of plant genes by using transgenic constructs that produce small RNAs in the plant.
Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger either RNA cleavage or translational inhibition of the target sequence. When bound to DNA target sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.
MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al., Science 294:853-858 (2001), Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al., Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001); Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., Genes. Dev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002); Reinhart et al., Genes. Dev 16:1616-1626 (2002)). They are processed from longer precursor transcripts that range in size from approximately 70 to 200 nt, and these precursor transcripts have the ability to form stable hairpin structures.
MicroRNAs (miRNAs) appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. It seems likely that miRNAs can enter at least two pathways of target gene regulation: (1) translational inhibition; and (2) RNA cleavage. MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nt short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants, and likely are incorporated into an RNA-induced silencing complex (RISC) that is similar or identical to that seen for RNAi.
A recombinant DNA construct (including a suppression DNA construct) of the present invention preferably comprises at least one regulatory sequence, such as a promoter.
A number of promoters can be used in recombinant DNA constructs of the present invention. The promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, inducible, or other promoters for expression in the host organism.
Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
High level, constitutive expression of the candidate gene under control of the 35S or UBI promoter may have pleiotropic effects, although candidate gene efficacy may be estimated when driven by a constitutive promoter. Use of tissue-specific and/or stress-specific promoters may eliminate undesirable effects but retain the ability to enhance alterations in plant architecture characteristics. This effect has been observed in Arabidopsis (Kasuga et al., (1999) Nature Biotechnol. 17:287-91).
Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
In choosing a promoter to use in the methods of the invention, it may be desirable to use a tissue-specific or developmentally regulated promoter.
A tissue-specific or developmentally regulated promoter is a DNA sequence that regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present invention that causes the desired temporal and spatial expression.
Promoters which are seed or embryo-specific and may be useful in the invention include soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)), patatin (potato tubers) (Rocha-Sosa, M., et al., (1989) EMBO J. 8:23-29), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al, (1991) Mol. Gen. Genet. 259:149-157; Newbigin, E. J., et al., (1990) Planta 180:461-470; Higgins, T. J. V., et al., (1988) Plant. Mol. Biol. 11:683-695), zein (maize endosperm) (Schemthaner, J. P., et al., (1988) EMBO J. 7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324), phytohemagglutinin (bean cotyledon) (Voelker, T. et al., (1987) EMBO J. 6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al., (1988) EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barley endosperm) (Marris, C., et al., (1988) Plant Mol. Biol. 10:359-366), glutenin and gliadin (wheat endosperm) (Colot, V., et al., (1987) EMBO J. 6:3559-3564), and sporamin (sweet potato tuberous root) (Hattori, T., et al., (1990) Plant Mol. Biol. 14:595-604). Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants. Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napes seeds (Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J 6:3559-3564 (1987)).
Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
Promoters include the following: 1) the stress-inducible RD29A promoter (Kasuga et al., (1999) Nature Biotechnol. 17:287-91); 2) the barley promoter, B22E; expression of B22E is specific to the pedicel in developing maize kernels (“Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers”. Klemsdal, S. S. et al., Mol. Gen. Genet. 228(112):9-16 (1991)); and 3) maize promoter, Zag2 (“Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS”, Schmidt, R. J. et al., Plant Cell 5(7):729-737 (1993); “Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUS-like MADS-box genes from maize”, Theissen et al., Gene 156(2):155-166 (1995); NCBI GenBank Accession No. X80206)). Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 days after pollination (“DAP”), and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP. Other useful promoters include any promoter that can be derived from a gene whose expression is maternally associated with developing female florets.
Additional preferred promoters for regulating the expression of the nucleotide sequences of the present invention in plants are stalk-specific promoters. Such stalk-specific promoters include the alfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al., Plant Mol. Biol. 27:513-528 (1995)) and S2B promoter (GenBank Accession No. EF030817) and the like, herein incorporated by reference.
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 comprise synthetic DNA segments. It is understood by those skilled in the art that 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. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B., Biochemistry of Plants 15:1-82 (1989).
Preferred promoters may include RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissue preferred promoters S2A (GenBank accession number EF030816) and S2B (GenBank accession number EF030817), and the constitutive promoter GOS2 from Zea mays. Other preferred promoters include root preferred promoters, such as the maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439, published Jul. 13, 2006), the maize ROOTMET2 promoter (WO05063998, published Jul. 14, 2005), the CR1BIO promoter (WO06055487, published May 26, 2006), the CRWAQ81 (WO05035770, published Apr. 21, 2005) and the maize ZRP2.47 promoter (NCBI accession number: U38790; GI No. 1063664),
Recombinant DNA constructs of the present invention may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences. In another preferred embodiment of the present invention, a recombinant DNA construct of the present invention further comprises an enhancer or silencer.
An intron sequence can be added to the 5′ untranslated region, the protein-coding region, or the 3′ untranslated region to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987).
Any plant can be selected for the identification of regulatory sequences and Squatty-Crinkle-Leaf polypeptide genes to be used in recombinant DNA constructs of the present invention. Examples of suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini. Particularly preferred plants for the identification of regulatory sequences are Arabidopsis, corn, wheat, soybean, and cotton.
A composition of the present invention is a plant comprising in its genome any of the recombinant DNA constructs (including any of the suppression DNA constructs) of the present invention (such as any of the constructs discussed above). Compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny, wherein the progeny or seed comprises within its genome the recombinant DNA construct (or suppression DNA construct). Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant. Progeny also includes hybrids and inbreds.
In hybrid seed propagated crops, mature transgenic plants can be self-pollinated to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced recombinant DNA construct (or suppression DNA construct). These seeds can be grown to produce plants that would exhibit an altered agronomic characteristic (e.g., an increased agronomic characteristic preferably under water limiting conditions), or used in a breeding program to produce hybrid seed, which can be grown to produce plants that would exhibit such an altered agronomic characteristic. The seeds may be maize seeds.
The plant may be a monocotyledonous or dicotyledonous plant, for example, a maize or soybean plant, such as a maize hybrid plant or a maize inbred plant. The plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane, or switchgrass.
The recombinant DNA construct may be stably integrated into the genome of the plant.
Particular embodiments include but are not limited to the following:
1. A plant (for example, a maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, and wherein said plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising said recombinant DNA construct. The plant may exhibit an alteration in a plant architecture characteristic selected from the group consisting of plant height, stalk length, internode length, leaf angle, leaf length, leaf surface, leaf width, leaf hair number, leaf hair volume, leaf initiation rate, leaf morphology, seedling size, and seedling growth rate.
2. A plant comprising in its genome a recombinant DNA construct comprising a suppression DNA construct comprising at least one regulatory element operably linked to: (i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52 or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising said recombinant DNA construct.
3. Any of the plants of the present invention wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane, and switchgrass.
4. A plant (for example, a maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a Squatty-Crinkle-Leaf polypeptide, and wherein said plant exhibits increased plant height when compared to a control plant not comprising said recombinant DNA construct. The plant may further exhibit an alteration in plant architecture when compared to the control plant.
The Squatty-Crinkle-Leaf polypeptide may be an ATP synthase D chain polypeptide.
5. A plant (for example, a maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a Squatty-Crinkle-Leaf polypeptide, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
6. A plant (for example, a maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
7. A plant (for example, a maize or soybean plant) comprising in its genome a suppression DNA construct comprising at least one regulatory element operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Squatty-Crinkle-Leaf polypeptide, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said suppression DNA construct.
8. A plant (for example, a maize or soybean plant) comprising in its genome a suppression DNA construct comprising at least one regulatory element operably linked to all or part of (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, or (b) a full complement of the nucleic acid sequence of (a), and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said suppression DNA construct.
9. Any progeny of the above plants in embodiments 1-8, any seeds of the above plants in embodiments 1-8, any seeds of progeny of the above plants in embodiments 1-8, and cells from any of the above plants in embodiments 1-6 and progeny thereof.
In any of the foregoing preferred embodiments 1-9 or any other embodiments of the present invention, the Squatty-Crinkle-Leaf polypeptide preferably is from Zea mays, Glycine max, Glycine tabacina, Glycine soja, Glycine tomentella, Arabidopsis thaliana, Oryza sativa, or Populus trichocarpa.
In any of the foregoing embodiments 1-9 or any other embodiments of the present invention, the recombinant DNA construct (or suppression DNA construct) may comprise at least a promoter functional in a plant as a regulatory sequence.
In any of the foregoing embodiments 1-9 or any other embodiments of the present invention, the alteration of at least one plant architecture characteristic is either an increase or decrease.
In any of the foregoing embodiments 1-9 or any other embodiments of the present invention, the at least one plant architecture characteristic may be selected from the group consisting of, but not limited to, plant height, stalk length, internode length, leaf angle, leaf length, leaf surface, leaf width, leaf hair number, leaf hair volume, leaf initiation rate, leaf morphology, seedling size, and seedling growth rate. For example, the alteration of at least one plant architecture characteristic may be an increase or decrease in plant height, a shorter leaf angle, an increase or decrease of internode length, an increase or decrease of leaf angle, and an increase or decrease of leaf width.
One of ordinary skill in the art would readily recognize a suitable control or reference plant to be utilized when assessing or measuring an alteration in at least one plant architecture characteristic or phenotype of a transgenic plant in any embodiment of the present invention in which a control or reference plant is utilized (e.g., compositions or methods as described herein). For example, by way of non-limiting illustrations:
1. Progeny of a transformed plant which is hemizygous with respect to a recombinant DNA construct (or suppression DNA construct), such that the progeny are segregating into plants either comprising or not comprising the recombinant DNA construct (or suppression DNA construct): the progeny comprising the recombinant DNA construct (or suppression DNA construct) would be typically measured relative to the progeny not comprising the recombinant DNA construct (or suppression DNA construct) (La, the progeny not comprising the recombinant DNA construct (or the suppression DNA construct) is the control or reference plant).
2. Introgression of a recombinant DNA construct (or suppression DNA construct) into an inbred line, such as in maize, or into a variety, such as in soybean: the introgressed line would typically be measured relative to the parent inbred or variety line (La, the parent inbred or variety line is the control or reference plant).
3. Two hybrid lines, where the first hybrid line is produced from two parent inbred lines, and the second hybrid line is produced from the same two parent inbred lines except that one of the parent inbred lines contains a recombinant DNA construct (or suppression DNA construct): the second hybrid line would typically be measured relative to the first hybrid line (i.e., the first hybrid line is the control or reference plant).
4. A plant comprising a recombinant DNA construct (or suppression DNA construct): the plant may be assessed or measured relative to a control plant not comprising the recombinant DNA construct (or suppression DNA construct) but otherwise having a comparable genetic background to the plant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of nuclear genetic material compared to the plant comprising the recombinant DNA construct (or suppression DNA construct)). There are many laboratory-based techniques available for the analysis, comparison and characterization of plant genetic backgrounds; among these are Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLP®s), and Simple Sequence Repeats (SSRs), which are also referred to as Microsatellites.
Furthermore, one of ordinary skill in the art would readily recognize that a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant would not include a plant that had been previously selected, via mutagenesis or transformation, for the desired agronomic characteristic or phenotype.
Methods include but are not limited to methods of altering at least one plant architecture characteristic in a plant, methods of determining an alteration of at least one plant architecture characteristic in a plant, methods of selecting maize plants or germplasm that display an alteration of at least one plant architecture characteristic, and methods of marker assisted selection. The plant may be a monocotyledonous or dicotyledonous plant, for example, a maize or soybean plant. The plant may also be maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane, or switchgrass. The seed may be a maize or soybean seed, for example, a maize hybrid seed or maize inbred seed.
Methods include but are not limited to the following:
A method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention. The cell transformed by this method is also included. In particular embodiments, the cell is a eukaryotic cell, e.g., a yeast, insect, or plant cell, or prokaryotic cell, e.g., a bacterial cell.
A method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides or recombinant DNA constructs (including suppression DNA constructs) of the present invention and regenerating a transgenic plant from the transformed plant cell. The invention is also directed to the transgenic plant produced by this method, and transgenic seed obtained from this transgenic plant. The transgenic plant obtained by this method may be used in other methods of the present invention.
A method for isolating a polypeptide of the invention from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the invention operably linked to at least one regulatory sequence, and wherein the transformed host cell is grown under conditions that are suitable for expression of the recombinant DNA construct.
A method of altering the level of expression of a polypeptide of the invention in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the polypeptide of the invention in the transformed host cell.
A method of the present invention includes a method of altering at least one plant architecture characteristic in a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) obtaining a progeny plant derived from the transgenic plant of step (b), wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits an alteration in at least one plant architecture characteristic when compared to a control plant not comprising the recombinant DNA construct.
A method of the present invention includes a method of altering at least one plant architecture characteristic in a plant, comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising (i) at least one regulatory sequence (for example, a promoter functional in a plant) operably linked to all or part of (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:39 or 52, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Squatty-Crinkle-Leaf polypeptide; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and (c) determining whether the transgenic plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising the suppression DNA construct. Optionally, said method further comprises: (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (e) determining whether the progeny plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising the suppression DNA construct.
A method of the present invention includes a method of determining an alteration of at least one plant architecture characteristic in a plant, comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:30 or 52; (b) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) determining whether the progeny plant exhibits an alteration of at least one plant architecture characteristic when compared to a control plant not comprising the recombinant DNA construct.
A method of the present invention includes a method of selecting a maize plant or germplasm that displays an alteration of at least one plant architecture characteristic comprising: a) obtaining DNA accessible for analysis; b) detecting the presence or absence of at least one allele of a marker locus comprising a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, c) selecting said maize plant or germplasm that comprises a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered.
A method of the present invention includes a method of selecting a maize plant or germplasm that displays an alteration of at least one plant architecture characteristic comprising: a) obtaining DNA accessible for analysis; b) detecting the presence or absence of at least one allele of a marker locus comprising a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, c) selecting said maize plant or germplasm that comprises a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered and wherein the at least one allele of the marker locus is located on a DNA interval between BAC c0137A18, or a nucleotide sequence that is 95% identical to BAC c0137A18 and BAC c0427D16, or a nucleotide sequence that is 95% identical to BAC c0427D16 based on the Clustal V method of alignment. Optionally, the at least one allele of the marker locus is on or within SEQ ID NO:39 or 52.
A method of the present invention includes a method of selecting a maize plant or germplasm that displays an altered plant architecture comprising: a) obtaining DNA accessible for analysis; b) detecting the presence of at least one allele of a first marker locus that is linked to and associated with an allele of a second marker locus, wherein the allele of the second marker locus comprises a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, c) selecting said maize plant or germplasm that comprises a point mutation at position 20 or 206, or both, of SEQ ID NO: 53;
A method of the present invention includes a method of marker assisted selection comprising: a) selecting a first maize plant that displays an alteration in at least one plant architecture characteristic comprising: i) obtaining DNA accessible for analysis; ii) detecting the presence of at least one allele of a first marker locus that is linked to and associated with an allele of a second marker locus, wherein the allele of the second marker locus comprises a mutation wherein base position 20 or 206, or both, of SEQ ID NO: 53 has been altered; and, iii) selecting said first maize plant that comprises a point mutation at position 20 or 206, or both, of SEQ ID NO: 53; b) crossing said first maize plant to a second maize plant; c) evaluating the progeny for at least said one allele of a first marker locus; and d) selecting progeny plants that possess at least said one allele of a first marker locus.
In any of the preceding methods or any other embodiments of methods of the present invention the plant can be selected from the group consisting of maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane, and switchgrass.
A method of the present invention includes a method of producing seed comprising any of the preceding methods, and further comprising obtaining seeds from said progeny plant, wherein said seeds comprise in their genome said recombinant DNA construct (or suppression DNA construct).
In any of the preceding methods or any other embodiments of methods of the present invention, in said introducing step said regenerable plant cell may comprise a callus cell, an embryogenic callus cell, a gametic cell, a meristematic cell, or a cell of an immature embryo. The regenerable plant cells may derive from an inbred maize plant. In any of the preceding preferred methods or any other embodiments of methods of the present invention, alternatives exist for introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence. For example, one may introduce into a regenerable plant cell a regulatory sequence (such as one or more enhancers, preferably as part of a transposable element), and then screen for an event in which the regulatory sequence is operably linked to an endogenous gene encoding a polypeptide of the instant invention.
The introduction of recombinant DNA constructs of the present invention into plants may be carried out by any suitable technique, including, but not limited to, direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation. Techniques for plant transformation and regeneration have been described in International Patent Publication WO 2009/006276, the contents of which are incorporated herein by reference in their entirety. The development or regeneration of plants containing the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in the art. The regenerated plants may be self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
Another embodiment of this invention includes genes that are differentially expressed in the SCL mutant versus the wild-type (such as those shown in Example 17).
The present invention is further illustrated in the following Examples, in which parts and percentages are by weight and degrees are in Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
To identify individual genes that affect maize plant architecture, a genetic approach by using EMS mutagenesis was developed. EMS mutagenesis was performed according to standard procedures (“Mutants of Maize” eds. M G Neuffer, E H Coe, S R Wessler, 1997, Cold Spring Harbor Laboratory Press, p. 397-398). The EMS mutagenized maize populations were screened for alterations in plant and organ growth. In short, the M1 families of the EMS mutagenized maize populations were grown in the greenhouse in 18-plant flats and approximately 500 flats of plants were grown and screened. The number of plants per family grown varied and depended upon the seed availability. Seedling plant architecture characteristics such as, but not limited to, leaf initiation rate, leaf morphology, seedling size, leaf angle, leaf length, and leaf width of mutant plants and wild type plants were observed at different stages during the germination and seedling growth.
Phenotypic changes were identified and monitored. At approximately v3 stage, mutant phenotypes became obvious and distinct from the wild type. As mutation of an individual gene is expected to be recessive in most cases, in the M1 family, only ¼ of the individual progeny is expected to be homozygous and show mutant phenotype and the rest is expected to show normal wild type phenotype. Mutants that fit approximately the segregation ratio were identified as a true mutation and advanced for further characterization.
Maize mutant seedlings were identified as having alterations in plant architecture such as reduced seedling size and shorter but wider leaf blades when compared to wild type (
The homozygote mutant plants were characterized by a semi-dwarf phenotype and by having a reduced plant height (
Two different alleles of the same gene from two independent mutations (SCL-338 and SCL-474,
Two recessive EMS mutants with similar phenotypes (SCL-474 and SCL-338) were identified from the EMS population described in Example 1 (PHN46 EMS population). Two large F2 (expected 75% wild and 25% mutant) populations were constructed by crossing homozygous mutant plants with a publicly available maize line A632. By genotyping 45 F2 mutant plants from SCL-338 and 53 mutant plants from SCL-474 with 81 SNP markers across the maize genome, both mutants were mapped in the same interval (chromosome 6 between PHM14535 (SEQ ID NO:1) at 90.31cM and PHM1147 (SEQ ID NO:4) at 120.91cM (Table 6)).
In order to fine map mutant genes, 259 and 275 F2 plants from SCL-474 and SCL-338, respectively, were grown in the greenhouse and genotyped. CAPS (Cleaved Amplified Polymorphic Site) markers were developed from the SCL interval for genotyping: PHM15457_F [SEQ ID NO: 10]; PHM15457_R [SEQ ID NO: 11] with restriction enzyme Hpall, and PHM4584_F [SEQ ID NO: 14]; PHM4584_R [SEQ ID NO: 15] with restriction enzyme Nsil. Both SCL-474 and SCL-338 mutants were mapped on chromosome 6 between PHM15457 (SEQ ID NO:2) at 90.4 cM and PHM4584 (SEQ ID NO:3) at 93.2 cM, implying that mutations in the same gene are responsible for the phenotypes of both SCL-338 and SCL-474.
To further fine map and clone the mutant genes, a SCL-338 F2 population with 2484 individuals was screened for recombinants. 1240 recombinants were identified from this F2 population between flanking markers PHM14535 (90.31 cM) and PHM1147 (120.91 cM). More markers were developed to genotype the recombinants: Indel (Insertion-deletion) marker c0137A18-B1-F[SEQ ID NO: 21], c0137A18-B1-R[SEQ ID NO: 22]; CAPS markers: c0427D16-D1_F [SEQ ID NO: 23] and c0427D16-D1-R [SEQ ID NO: 24] with restriction enzyme Fokl; c0427D16-A1-F [SEQ ID NO: 25] and c0427D16-A1-R [SEQ ID NO: 26] with restriction enzyme BsiEl; PHM589962-3-F[SEQ ID NO: 27] and PHM589962-3-R[SEQ ID NO: 28] with restriction enzyme Mnll; PHM589962-4-F[SEQ ID NO: 29] and PHM589962-4-R[SEQ ID NO: 30] with restriction enzyme Mwol. These markers and recombinants enable the SCL-338 mutant be mapped within a 2 BAC interval (bac c0137A18 and bac c0427D16), defined by c0137A18-B1 and c0427D16-D1 (with 1 recombinant on each side). More CAPS markers were developed within this 2 BAC interval but failed to narrow down the region further due to the lack of recombinants.
CAPS marker amplifications were performed in a 10 ul PCR reaction using the Qiagen HotStart mix and 15 ng DNA. The PCR program was: 94° C. for 14 min (1 cycle); 94° C. for 60 sec, 55° C. for 60 sec, and 72° C. for 60 sec, (35 cycles); and 72° C. for 7 min. 10 ul of the amplification product was used for a restriction digest (total volume of 20 ul) with the appropriate restriction enzymes. Restriction reactions were carried out at the recommended temperature for six hours. Restricted amplification products were examined on 2% agarose gels.
In order to identify the candidate gene for SCL-338 mutant, genes predicted by FGENESH (Salamov, A. and Solovyev, V. (2000) Genome Res., 10: 516-522) within the 2-BAC interval (BAC c0137A18 and BAC c0427D16) were identified and sequence compared between Hg11 and the SCL-338 mutant.
A point mutation at base number 2105 of SEQ ID NO: 31 (G to A) at an exon-intron junction of an AP2-like gene was identified in the SCL-338 mutant. Interestingly, in SCL-474, a different point mutation at base number 1919 of SEQ ID NO: 31 (G to A) near another exon-intron junction was detected. This implies that both SCL-338 and SCL-474 phenotypes are caused by mutations within the same gene, and both mutant alleles may affect RNA splicing. An alignment of a fragment of the genomic DNA sequence surrounding the base deletions of Wild type maize (SEQ ID NO:31) is shown in
To confirm SCL-338 and SCL-474 are allelic, several heterozygous SCL-474 and SCL-338 were reciprocally crossed and 5 F1 ears were generated. Seventy-two plants from each F1 ear were phenotyped for progeny test. Mutant phenotypes were observed in all F1 progenies and the ratio between wild and mutant is close to 3:1. This data support the conclusion that SCL-474 and SCL-339 are two alleles of the same gene and mutations in the AP2-like gene cause the mutant phenotypes.
Primers CDS1-F [SEQ ID NO: 34] and CDS1-R [SEQ ID NO: 35] were designed to span the exons around the mutations for RT-PCR. Size difference in cDNA was observed between wild type and SCL-338 and the cDNA fragments were cloned.
Sequencing of 125 SCL-338 mutants and 95 SCL-474 mutant clones (Table 7) showed that mis-spliced molecules represent the predominant form in mutant plants (98.4% and 90.8% in SCL-338 and SCL-474, respectively).
RT-PCR as well as 5′ and 3′ RACE were performed to generate the full length cDNA sequence for the AP2-like gene. Total RNA was extracted from wild type and two homozygous mutants' mature leaves using a Qiagen RNeasy kit and cDNA obtained with oligo DT and Superscript® reverse transcriptase (Invitrogen). PCR was performed and PCR products were sequenced. 3′ and 5′ RACE were performed to identify 3′ and 5′ UTR.
The SCL gene's genomic structure was determined by aligning the CDS sequence (SEQ ID NO: 36) with the genomic sequence (SEQ ID NO:31). The SCL candidate gene consists of 9 exons and 8 introns (Table 8).
An alignment of the amino acid sequences encoded by the dominant cDNAs of wild type maize (SEQ ID NO: 39) and mutant maize (SCL338, SEQ ID NO:49 and SCL-474, SEQ ID NO:50) is shown in
The sequence of the SCL genomic DNA (SEQ ID NO: 31) or cDNA (SEQ ID NO:36) encoded a putative polypeptide of 412 amino acids (SEQ ID NO: 39). A homology search of this protein revealed that SCL is an AP2-like transcription factor (
The expression pattern of the SCL gene was examined using Massively Parallel Signature Sequencing (MPSS; Lynx Therapeutics, Berkeley, USA). Briefly, cDNA libraries were constructed and immobilized on microbeads as described in Brenner et al., (2000) Nat. Biotechnol. 18(6): 630-634. The construction of the library on a solid support allows the library to be arrayed in a monolayer and thousands of clones to be subjected to nucleotide sequence analysis in parallel. The analysis results in a “signature” 17-mer sequence whose frequency of occurrence is proportional to the abundance of that transcript in the plant tissue. A 17-mer unique tag (Table 9) positioned at the last exon close to 3′UTR region of the SCL gene was identified. The SCL gene is expressed in almost all the tissues, with the tassel tissue showing the highest expression level (
Destination vector PHP23236 (
cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in UNI-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The UNI-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene.
Upon conversion, cDNA inserts will be contained in the plasmid vector pBLUESCRIPT®. In addition, the cDNAs may be introduced directly into precut pBLUESCRIPT® II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DF-110B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBLUESCRIPT® plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for F1S are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers.
Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.
Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (GIBCO BRL/Life Technologies, Rockville, Md.) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI PRISM® dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.
Sequence data is collected (ABI PRISM® Collections) and assembled using Phred and Phrap (Ewing et al., (1998) Genome Res. 8:175-185; Ewing and Green (1998) Genome Res. 8:186-194). Phred is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (Gordon et al., (1998) Genome Res. 8:195-202).
In some of the clones, the cDNA fragment may correspond to a portion of the 3′-terminus of the gene and does not cover the entire open reading frame. In order to obtain the upstream information, one of two different protocols is used. The first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame. Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries sometimes are chosen based on previous knowledge that the specific gene should be found in a certain tissue and sometimes are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries. Library pools are normally prepared using from 3 to 5 different libraries and are normalized to a uniform dilution. In the first round of amplification both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5′-terminus of the clone coupled with a gene-specific (reverse) primer. The first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3′-untranslated region (also referred to as UTR). In the second round of amplification, a nested set of primers is used for both methods. The resulting DNA fragment is ligated into a pBLUESCRIPT® vector using a commercial kit and following the manufacturer's protocol. This kit is selected from many available from several vendors including INVITROGEN™ (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and GIBCO-BRL (Gaithersburg, Md.). The plasmid DNA is isolated by the alkaline lysis method and is submitted for sequencing and assembly using Phred/Phrap, as above.
Using the INVITROGEN™ GATEWAY® LR Recombination technology, the protein coding region, of the maize SCL gene from clone p0031.ccmau15r:fis, was directionally cloned into the destination vector PHP29634 (SEQ ID NO:46) to create an expression vector, PHP35056. The SCL gene present in clone p0031.ccmau15r:fis (SEQ ID NO:50) encodes an SCL polypeptide (SEQ ID NO: 52) which constitutes a variant of SEQ ID NO:39 (
Maize plants can be transformed to overexpress the Arabidopsis lead gene or the corresponding homologs from other species in order to examine the resulting phenotype.
Recipient Plants:
Recipient plant cells can be from a uniform maize line having a short life cycle (“fast cycling”), a reduced size, and high transformation potential. Typical of these plant cells for maize are plant cells from any of the publicly available Gaspe Flint (GBF) line varieties. One possible candidate plant line variety is the F1 hybrid of GBF×QTM (Quick Turnaround Maize, a publicly available form of Gaspe Flint selected for growth under greenhouse conditions) disclosed in Tomes et al., U.S. Patent Application Publication No. 2003/0221212. Transgenic plants obtained from this line are of such a reduced size that they can be grown in four-inch pots (¼ the space needed for a normal sized maize plant) and mature in less than 2.5 months. (Traditionally 3.5 months is required to obtain transgenic T0 seed once the transgenic plants are acclimated to the greenhouse.) Another suitable line is a double haploid line of GS3 (a highly transformable line) X Gaspe Flint. Yet another suitable line is a transformable elite inbred line carrying a transgene that causes early flowering, reduced stature, or both.
Transformation Protocol:
Any suitable method may be used to introduce the transgenes into the maize cells, including, but not limited to, inoculation type procedures using Agrobacterium based vectors. Transformation may be performed on immature embryos of the recipient (target) plant.
Precision Growth and Plant Tracking:
The event population of transgenic (T0) plants resulting from the transformed maize embryos is grown in a controlled greenhouse environment using a modified randomized block design to reduce or eliminate environmental error. A randomized block design is a plant layout in which the experimental plants are divided into groups (e.g., thirty plants per group), referred to as blocks, and each plant is randomly assigned a location within the block.
For a group of thirty plants, twenty-four transformed, experimental plants and six control plants (plants with a set phenotype) (collectively, a “replicate group”) are placed in pots which are arranged in an array (a.k.a., a replicate group or block) on a table located inside a greenhouse. Each plant, control or experimental, is randomly assigned to a location with the block which is mapped to a unique, physical greenhouse location as well as to the replicate group. Multiple replicate groups of thirty plants each may be grown in the same greenhouse in a single experiment. The layout (arrangement) of the replicate groups should be determined to minimize space requirements as well as environmental effects within the greenhouse. Such a layout may be referred to as a compressed greenhouse layout.
An alternative to the addition of a specific control group is to identify those transgenic plants that do not express the gene of interest. A variety of techniques such as RT-PCR can be applied to quantitatively assess the expression level of the introduced gene. T0 plants that do not express the transgene can be compared to those which do.
Each plant in the event population is identified and tracked throughout the evaluation process, and the data gathered from that plant is automatically associated with that plant so that the gathered data can be associated with the transgene carried by the plant. For example, each plant container can have a machine-readable label (such as a Universal Product Code (UPC) bar code) which includes information about the plant identity, which in turn is correlated to a greenhouse location so that data obtained from the plant can be automatically associated with that plant.
Alternatively any efficient, machine readable, plant identification system can be used, such as two-dimensional matrix codes or even radio frequency identification tags (RFID) in which the data is received and interpreted by a radio frequency receiver/processor. See U.S. Published Patent Application No. 2004/0122592, incorporated herein by reference.
Phenotypic Analysis Using Three-Dimensional Imaging:
Each greenhouse plant in the T0 event population, including any control plants, is analyzed for agronomic characteristics of interest, and the agronomic data for each plant is recorded or stored in a manner so that it is associated with the identifying data (see above) for that plant. Confirmation of a phenotype (gene effect) can be accomplished in the T1 generation with a similar experimental design to that described above.
The T0 plants are analyzed at the phenotypic level using quantitative, non-destructive imaging technology throughout the plant's entire greenhouse life cycle to assess the traits of interest. A digital imaging analyzer may be used for automatic multi-dimensional analyzing of total plants. The imaging may be done inside the greenhouse. Two camera systems, located at the top and side, and an apparatus to rotate the plant, are used to view and image plants from all sides. Images are acquired from the top, front and side of each plant. All three images together provide sufficient information to evaluate the biomass, size and morphology of each plant.
Due to the change in size of the plants from the time the first leaf appears from the soil to the time the plants are at the end of their development, the early stages of plant development are best documented with a higher magnification from the top. This may be accomplished by using a motorized zoom lens system that is fully controlled by the imaging software.
In a single imaging analysis operation, the following events occur: (1) the plant is conveyed inside the analyzer area, rotated 360 degrees so its machine readable label can be read, and left at rest until its leaves stop moving; (2) the side image is taken and entered into a database; (3) the plant is rotated 90 degrees and again left at rest until its leaves stop moving, and (4) the plant is transported out of the analyzer,
Plants are allowed at least six hours of darkness per twenty-four hour period in order to have a normal day/night cycle.
Imaging Instrumentation:
Any suitable imaging instrumentation may be used, including but not limited to light spectrum digital imaging instrumentation commercially available from LemnaTec GmbH of Wurselen, Germany. The images are taken and analyzed with a LemnaTec Scanalyzer HTS LT-0001-2 having a ½″ IT Progressive Scan IEE CCD imaging device. The imaging cameras may be equipped with a motor zoom, motor aperture and motor focus. All camera settings may be made using LemnaTec software. For example, the instrumental variance of the imaging analyzer is less than about 5% for major components and less than about 10% for minor components.
Software:
The imaging analysis system comprises a LemnaTec HTS Bonit software program for color and architecture analysis and a server database for storing data from about 500,000 analyses, including the analysis dates. The original images and the analyzed images are stored together to allow the user to do as much reanalyzing as desired. The database can be connected to the imaging hardware for automatic data collection and storage. A variety of commercially available software systems (e.g., Matlab, others) can be used for quantitative interpretation of the imaging data, and any of these software systems can be applied to the image data set.
Conveyor System:
A conveyor system with a plant rotating device may be used to transport the plants to the imaging area and rotate them during imaging. For example, up to four plants, each with a maximum height of 1.5 m, are loaded onto cars that travel over the circulating conveyor system and through the imaging measurement area. In this case, the total footprint of the unit (imaging analyzer and conveyor loop) is about 5 m×5 m.
The conveyor system can be enlarged to accommodate more plants at a time. The plants are transported along the conveyor loop to the imaging area and are analyzed for up to 50 seconds per plant. Three views of the plant are taken. The conveyor system, as well as the imaging equipment, should be capable of being used in greenhouse environmental conditions.
Illumination:
Any suitable mode of illumination may be used for the image acquisition. For example, a top light above a black background can be used. Alternatively, a combination of top- and backlight using a white background can be used. The illuminated area should be housed to ensure constant illumination conditions. The housing should be longer than the measurement area so that constant light conditions prevail without requiring the opening and closing or doors. Alternatively, the illumination can be varied to cause excitation of either transgene (e.g., green fluorescent protein (GFP), red fluorescent protein (REP)) or endogenous (e.g., Chlorophyll) fluorophores.
Biomass Estimation Based on Three-Dimensional Imaging:
For best estimation of biomass, the plant images should be taken from at least three axes, for example, the top and two side (sides 1 and 2) views. These images are then analyzed to separate the plant from the background, pot, and pollen control bag (if applicable). The volume of the plant can be estimated by the calculation:
In the equation above, the units of volume and area are “arbitrary units.” Arbitrary units are entirely sufficient to detect gene effects on plant size and growth in this system because what is desired is to detect differences (both positive-larger and negative-smaller) from the experimental mean, or control mean. The arbitrary units of size (e.g., area) may be trivially converted to physical measurements by the addition of a physical reference to the imaging process. For instance, a physical reference of known area can be included in both top and side imaging processes. Based on the area of these physical references a conversion factor can be determined to allow conversion from pixels to a unit of area, such as square centimeters (cm2). The physical reference may or may not be an independent sample. For instance, the pot, with a known diameter and height, could serve as an adequate physical reference.
Color Classification:
The imaging technology may also be used to determine plant color and to assign plant colors to various color classes. The assignment of image colors to color classes is an inherent feature of the LemnaTec software. With other image analysis software systems, color classification may be determined by a variety of computational approaches.
For the determination of plant size and growth parameters, a useful classification scheme is to define a simple color scheme including two or three shades of green and, in addition, a color class for chlorosis, necrosis and bleaching, should these conditions occur. A background color class which includes non-plant colors in the image (for example pot and soil colors) is also used and these pixels are specifically excluded from the determination of size. The plants are analyzed under controlled constant illumination so that any change within one plant over time, or between plants or different batches of plants (e.g., seasonal differences) can be quantified.
In addition to its usefulness in determining plant size growth, color classification can be used to assess other yield component traits. For these other yield component traits additional color classification schemes may be used. For instance, the trait known as “staygreen,” which has been associated with improvements in yield, may be assessed by a color classification that separates shades of green from shades of yellow and brown (which are indicative of senescing tissues). By applying this color classification to images taken toward the end of the T0 or T1 plants' life cycle, plants that have increased amounts of green colors relative to yellow and brown colors (expressed, for instance, as Green/Yellow Ratio) may be identified. Plants with a significant difference in this Green/Yellow ratio can be identified as carrying transgenes that impact this important agronomic trait.
The skilled plant biologist will recognize that other plant colors arise which can indicate plant health or stress response (for instance anthocyanins), and that other color classification schemes can provide further measures of gene action in traits related to these responses.
Plant Architecture Analysis:
Transgenes which modify plant architecture parameters may also be identified using the present invention, including such parameters as maximum height and width, internodal distances, angle between leaves and stem, number of leaves starting at nodes, and leaf length. The LemnaTec system software may be used to determine plant architecture as follows. The plant is reduced to its main geometric architecture in a first imaging step and then, based on this image, parameterized identification of the different architecture parameters can be performed. Transgenes that modify any of these architecture parameters either singly or in combination can be identified by applying the statistical approaches previously described.
Pollen Shed Date:
Pollen shed date is an important parameter to be analyzed in a transformed plant, and may be determined by the first appearance on the plant of an active male flower. To find the male flower object, the upper end of the stem is classified by color to detect yellow or violet anthers. This color classification analysis is then used to define an active flower, which in turn can be used to calculate pollen shed date.
Alternatively, pollen shed date and other easily visually detected plant attributes (e.g., pollination date, first silk date) can be recorded by the personnel responsible for performing plant care. To maximize data integrity and process efficiency this data is tracked by utilizing the same barcodes utilized by the LemnaTec light spectrum digital analyzing device. A computer with a barcode reader, a palm device, or a notebook PC may be used for ease of data capture recording time of observation, plant identifier, and the operator who captured the data.
Orientation of the Plants:
Mature maize plants grown at densities approximating commercial planting often have a planar architecture. That is, the plant has a clearly discernable broad side, and a narrow side. The image of the plant from the broadside is determined. To each plant, a well-defined basic orientation is assigned to obtain the maximum difference between the broadside and edgewise images. The top image is used to determine the main axis of the plant, and an additional rotating device is used to turn the plant to the appropriate orientation prior to starting the main image acquisition.
Sequences homologous to the maize SCL polypeptide can be identified using sequence comparison algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health). Sequences encoding homologous SCL polypeptides can be PCR-amplified by any of the following methods.
Method 1 (RNA-based): If the 5′ and 3′ sequence information for the protein-coding region of a gene encoding a SCL polypeptide homolog is available, gene-specific primers can be designed as outlined in Example 5. RT-PCR can be used with plant RNA to obtain a nucleic acid fragment containing the protein-coding region flanked by attB1 and attB2 sequences. The primer may contain a consensus Kozak sequence (CAACA) upstream of the start codon.
Method 2 (DNA-based): Alternatively, if a cDNA clone is available for a gene encoding a SCL polypeptide, the entire cDNA insert (containing 5′ and 3′ non-coding regions) can be PCR amplified. Forward and reverse primers can be designed that contain either the attB1 sequence and vector-specific sequence that precedes the cDNA insert or the attB2 sequence and vector-specific sequence that follows the cDNA insert, respectively.
Method 3 (genomic DNA): Genomic sequences can be obtained using long range genomic PCR capture. Primers can be designed based on the sequence of the genomic locus and the resulting PCR product can be sequenced. The sequence can be analyzed using the FGENESH (Salamov, A. and Solovyev, V. (2000) Genome Res., 10: 516-522) program, and optionally, can be aligned with homologous sequences from other species to assist in identification of putative introns.
Methods 1, 2, and 3 can be modified according to procedures known by one skilled in the art. For example, the primers of Method 1 may contain restriction sites instead of attB1 and attB2 sites, for subsequent cloning of the PCR product into a vector containing attB1 and attB2 sites. Additionally, Method 2 can involve amplification from a cDNA clone, a lambda clone, a BAC clone or genomic DNA.
A PCR product obtained by either method above can be combined with the GATEWAY® donor vector, using a BP Recombination Reaction. This process removes the bacteria lethal ccdB gene, as well as the chloramphenicol resistance gene (CAM) from pDONRTM221 and directionally clones the PCR product with flanking attB1 and attB2 sites to create an entry clone. Using the INVITROGEN™ GATEWAY® CLONASETM technology, the sequence encoding the homologous SCL polypeptide, from the entry clone can then be transferred to a suitable destination vector to obtain a plant expression vector for use with Arabidopsis, soybean, or corn.
Alternatively, a MultiSite GATEWAY® LR recombination reaction between multiple entry clones and a suitable destination vector can be performed to create an expression vector.
Soybean plants can be transformed to overexpress a SCL gene or the corresponding homologs from various species in order to examine the resulting phenotype.
The SCL gene or SCL homolog can be directionally cloned using the INVITROGEN™ GATEWAY® CLONASE™ technology such that expression of the gene is under control of the SCP1 promoter.
Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. Techniques for soybean transformation and regeneration have been described in International Patent Publication WO 20091006276, the contents of which are herein incorporated by reference.
T1 plants can be analyzed for alterations in plant architecture characteristics as described in Example 1.
Maize plants can be transformed to overexpress or silence an SCL gene or the corresponding homologs from various species in order to examine the resulting phenotype.
Using the INVITROGEN™ GATEWAY® CLONASE™ technology, the SCL gene can be directionally cloned into a maize transformation vector. Expression of the gene in the maize transformation vector can be under control of a constitutive promoter such as the maize ubiquitin promoter (Christensen et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen et al., (1992) Plant Mal. Biol. 18:675-689) or under the control of a tissue specific promoter.
The recombinant DNA construct described above can then be introduced into corn cells by particle bombardment. Techniques for corn transformation by particle bombardment have been described in International Patent Publication WO 20091006276, the contents of which are herein incorporated by reference.
T1 plants can be analyzed for alterations in plant architecture characteristics as described in Example 1.
Electroporation competent cells (40 μL), such as Agrobacterium tumefaciens LBA4404 containing PHP10523 (
Aliquots of 250 pt are spread onto plates containing YM medium and 50 μg/mL spectinomycin and incubated three days at 28-30° C. To increase the number of transformants one of two optional steps can be performed:
Option 1: Overlay plates with 30 μL of 15 mg/mL rifampicin. LBA4404 has a chromosomal resistance gene for rifampicin. This additional selection eliminates some contaminating colonies observed when using poorer preparations of LBA4404 competent cells.
Option 2: Perform two replicates of the electroporation to compensate for poorer electrocompetent cells.
Identification of Transformants:
Four independent colonies are picked and streaked on plates containing AB minimal medium and 50 μg/mL spectinomycin for isolation of single colonies. The plates are incubated at 28° C. for two to three days. A single colony for each putative co-integrate is picked and inoculated with 4 mL of 10 g/L bactopeptone, 10 g/L yeast extract, 5 g/L sodium chloride and 50 mg/L spectinomycin. The mixture is incubated for 24 h at 28° C. with shaking. Plasmid DNA from 4 mL of culture is isolated using a Qiagen® Miniprep and an optional Buffer PB wash. The DNA is eluted in 304. Aliquots of 2 μL are used to electroporate 20 μL of DH10b+20 μL of twice distilled H2O as per above. Optionally a 154 aliquot can be used to transform 75-100 μL of INVITROGEN™ Library Efficiency DH5a. The cells are spread on plates containing LB medium and 50 μg/mL spectinomycin and incubated at 37° C. overnight.
Three to four independent colonies are picked for each putative co-integrate and inoculated into 4 mL of 2xYT medium (10 g/L bactopeptone, 10 g/L yeast extract, 5 g/L sodium chloride) with 50 μg/mL spectinomycin. The cells are incubated at 37° C. overnight with shaking. Next, isolate the plasmid DNA from 4 mL of culture using QIAprep® Miniprep with optional Buffer PB wash (elute in 50 μL). Use 8 μL for digestion with SaII (using parental DNA and PHP10523 as controls). Three more digestions using restriction enzymes BamHI, EcoRI, and HindIII are performed for 4 plasmids that represent 2 putative co-integrates with correct Sall digestion pattern (using parental DNA and PHP10523 as controls). Electronic gels are recommended for comparison.
Maize plants can be transformed to overexpress or silence a SCL gene or the corresponding homologs from various species in order to examine the desired phenotype.
Agrobacterium-mediated transformation of maize is performed essentially as described by Zhao et al., in Meth. Mol. Biol. 318:315-323 (2006) (see also Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No. 5,981,840 issued Nov. 9, 1999, incorporated herein by reference). The transformation process involves bacterium inoculation, co-cultivation, resting, selection, and plant regeneration.
1. Immature Embryo Preparation:
Immature maize embryos are dissected from caryopses and placed in a 2 mL microtube containing 2 mL PHI-A medium.
2. Agrobacterium Infection and Co-Cultivation of Immature Embryos:
2.1 Infection Step:
PHI-A medium of (1) is removed with 1 mL micropipettor, and 1 mL of Agrobacterium suspension is added. The tube is gently inverted to mix. The mixture is incubated for 5 min at room temperature.
2.2 Co-culture Step:
The Agrobacterium suspension is removed from the infection step with a 1 mL micropipettor. Using a sterile spatula, the embryos are scraped from the tube and transferred to a plate of PHI-B medium in a 100×15 mm Petri dish. The embryos are oriented with the embryonic axis down on the surface of the medium. Plates with the embryos are cultured at 20° C., in darkness, for three days. L-Cysteine can be used in the co-cultivation phase. With the standard binary vector, the co-cultivation medium supplied with 100-400 mg/L L-cysteine is critical for recovering stable transgenic events.
3. Selection of Putative Transgenic Events:
To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos are transferred, maintaining orientation and the dishes are sealed with parafilm. The plates are incubated in darkness at 28° C. Actively growing putative events, as pale yellow embryonic tissue, are expected to be visible in six to eight weeks. Embryos that produce no events may be brown and necrotic, and little friable tissue growth is evident. Putative transgenic embryonic tissue is subcultured to fresh PHI-D plates at two-three week intervals, depending on growth rate. The events are recorded.
a. Regeneration of T0 Plants:
Embryonic tissue propagated on PHI-D medium is subcultured to PHI-E medium (somatic embryo maturation medium), in 100×25 mm Petri dishes and incubated at 28° C., in darkness, until somatic embryos mature, for about ten to eighteen days. Individual, matured somatic embryos with well-defined scutellum and coleoptile are transferred to PHI-F embryo germination medium and incubated at 28° C. in the light (about 80 μE from cool white or equivalent fluorescent lamps). In seven to ten days, regenerated plants, about 10 cm tall, are potted in horticultural mix and hardened-off using standard horticultural methods.
Media for Plant Transformation:
4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos (filter-sterilized).
5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL 11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCI, 0.5 mg/L pyridoxine HCI, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5 mg/L zeatin (Sigma, Cat. No. Z-0164), 1 mg/L indole acetic acid (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L bialaphos (filter-sterilized), 100 mg/L carbenicillin (filter-sterilized), 8 g/L agar, pH 5.6.
Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks, the tissue can be transferred to regeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).
Transgenic T0 plants can be regenerated and their phenotype determined. T1 seed can be collected.
Furthermore, a recombinant DNA construct containing a validated Arabidopsis gene can be introduced into an elite maize inbred line either by direct transformation or introgression from a separately transformed line.
Transgenic plants, either inbred or hybrid, can undergo more vigorous field-based experiments to study alteration in plant architecture.
Preparation of SCL Gene Expression Vector for Transformation of Maize Using INVITROGEN's™ GATEWAY® technology, an LR Recombination Reaction can be performed with an entry clone containing the SCL gene and a destination vector to create a precursor plasmid (ATPQ-precursor). The precursor plasmid can contain the following expression cassettes:
1. Ubiquitin promoter::moPAT::PinII terminator; cassette expressing the PAT herbicide resistance gene used for selection during the transformation process.
2. LTP2 promoter::DS-RED2::PinII terminator; cassette expressing the DS-RED color marker gene used for seed sorting.
3. Ubiquitin promoter:SCL:PinII terminator; cassette overexpressing the gene of interest encoding the SCL polypeptide.
A full length codon sequence of the SCL variant present in clone p0031.ccmau15r:fis was used to generate over-expression transgenic plants as described in the previous examples. Two T2 families (18 plants/family) were selected for phenotyping based on SCL expression levels and seed availability. Significant difference in plant height between transgenic plants and nulls (control plants not containing the transgene) was observed in transgenic events Trans4, Trans5 and Trans6 (Table 10).
Wild type (WT) and mutant (SCL) maize plants were analyzed at the seedling and mature stage. One seedling each at the V3 stage, one mature plant each at the stage just after flowering.
Leaf samples were collected by cutting a 2 cm wide strip from the mid-point of leaf #2 from V3 seedlings and from leaf #5 from mature plants. Samples were fixed in a solution of 25% acetic acid and 75% ethanol. Samples were further processed by taking 6 mm leaf punches from corresponding regions of fixed tissue and post-fixing in 2% glutaraldehyde to enhance host cell autofluorescence. These post-fixed leaf disks were rinsed, cleared in chloral hydrate, mounted in Hoyer's medium, and examined with the 720 nm laser line of a Zeiss multiphoton laser scanning microscope, using a 20× Plan Apochromat (0.75 NA) objective lens. Multiple optical sections (0.8 μm section thickness) were collected and maximum intensity projections assembled as single images for evaluation. The V3 stage leaf epidermis is shown in
Maize stalk samples were collected by cutting 2 cm wide cross-sections of stalk from the center of internode #3 (base) and #9 (apex) and fixing in acetic acid-ethanol. Cross-sections were post-fixed in glutaraldehyde to enhance cell wall autofluorescence, cleared in chloral hydrate, mounted, and examined with a multiphoton laser scanning microscope (LSM). Post-flowering maize stalk upper internode (apex) of wild type and SCL mutant maize plants are shown in
Microarray experiments were conducted on V3 seedling (V3-SDL), V8 leaf (V8-LF), and V8 stalk (V8-STK) with 3 replicates to identify differentially expressed genes between SCL mutant and wild type plants. Each replicate contains at least 5 wild type or homozygous mutant F2 plants (PHN46-SCL—338/A632), respectively. Using the criteria of fold change ≧1.8 and P-value ≦0.0001, all the differentially expressed genes were selected for further analysis. In summary, 1068 genes were differentially expressed in V3-SDL, among which 548 genes were up regulated and 520 genes were down regulated in SCL mutants. 3401 genes were differentially expressed in V8-LF, among which 1816 genes were up regulated and 1585 genes were down regulated in SCL mutants. 3305 genes were differentially expressed in V8-STK, among which 1852 genes were up regulated and 1453 genes were down regulated in SCL mutants.
Since all the mutant F2 plants have the PHN46 genomic segments near the SCL locus, while all the wild type plants have the A632 allele at the SCL locus, the differentially expressed genes mapped near the SCL locus on chromosome 6 are likely the results of genotype variations and not caused by the SCL mutation. The number of differentially expressed genes remaining after all the genes mapped on chromosome 6 are eliminated from the list is also listed in Table 11. The accession number in the following Tables corresponds to the accession number from NCBI (National Center for Biotechnology Information). All the analyses below are based on the differentially expressed genes not mapped on chromosome 6.
Interestingly, a lot of plant hormone related genes were found to be differentially expressed. The 40 kDa PI 8.5 ABSCISSIC acid-induced is found to be up regulated in V3 mutant seedling (Table 12). IAA1 protein, gibberellin 20-oxidase, auxin induced protein, putative ABA response element binding factor, cytokinin oxidase 2, AUX1 protein, and gibberellin 2-oxidase are found to be down regulated, yet auxin efflux carrier family protein-like, indole-3-glycerol phosphate lyase (chloroplast precursor), putative brassinosteroid insensitive 1, putative gibberellin 20-oxidase, (+)-abscisic acid 8-hydroxylase, 40 kDa PI 8.5 ABSCISSIC acid-induced protein, and putative auxin-regulated protein are up regulated in V8 mutant leaf (Table 13 and Table 14). Auxin-induced protein-related-like protein, auxin induced protein, gibberellin 2-oxidase, cytokinin oxidase 3, gibberellin 2-oxidase, ethylene-responsive factor-like protein 1, auxin-induced protein-like, and GA 3-oxidase 2 are found to be up regulated, yet putative auxin-induced protein family, auxin-induced protein-like, indole-3-glycerol phosphate lyase, chloroplast precursor, putative indole-3-glycerol phosphate synthase, putative ABA response element binding factor, and putative ethylene-inducible CTR1-like protein kinase are down regulated in V8 mutant stalk (Table 15 and Table 16).
Among those genes, auxin induced protein, gibberellin 2-oxidase, and cytokinin oxidase 3 are found to be down regulated, and indole-3-glycerol phosphate lyase is found to be up regulated in both leaf and stalk of V8 mutant plants. Putative ABA response element binding factor is found to be down regulated in V8 mutant leaf, yet up regulated in V8 mutant stalk (Table 17, 18, 19).
To identify the pathway that may be affected by the SCL gene, the microarray data was further analyzed by Pathway Studio software (version 7), developed by Ariadne Genomics, for GO ontology and sub-network Enrichment Analysis (Broad Institute; PNAS_Oct. 25, 2005_vol. 102_no. 43—15549) using Fisher exact Test (Fisher, R. A. (1922). “On the interpretation of χ2 from contingency tables, and the calculation of P”. Journal of the Royal Statistical Society 85 (1): 87-94. doi:10.2307/2340521. JSTOR 2340521.Fisher, R. A. (1954). Statistical Methods for Research Workers. Oliver and Boyd.) at P<=0.05 threshold. The enriched pathways found through those analyses are likely directly or indirectly regulated by SCL.
Among those genes that are differentially expressed between mutants and wild plants, genes related to response to thiol-disulfide exchange intermediate activity, translation, embryonic development ending in seed dormancy, response to gibberellin stimulus, structural constituent of ribosome, and intracellular are enriched in V8-LF. The P values are 0.00272478, 0.0127758, 0.0157532, 0.0186005, 0.025365, and 0.0449772, respectively. The corresponding GO categories are molecular function, biological process, biological process, biological process, molecular function, and cellular component, respectively (Table 21). Genes related to auxin polar transport, response to gibberellin stimulus, UDP-glycosyltransferase activity, protein folding, lipid metabolic process, hydrolase activity, ATPase activity coupled to transmembrane movement of substances, protein amino acid dephosphorylation, and response to ethylene stimulus are enriched in V8-STK. The P values are 0.004714, 0.004769, 0.006713, 0.007205, 0.007539, 0.007903, 0.008138, 0.008294, and 0.009463, respectively. GO categories are biological process, biological process, molecular function, biological process, biological process, molecular function, molecular function, biological process, and biological process, respectively (Table 22). Sub-Network Enrichment Analysis Fisher Exact Test indicates that genes related to jasmonic acid, methyl jasmonate, cycloheximide, salicylic acid, and ethylene are enriched in V3-SDL. The P values are 1.95E-06, 7.03E-05, 0.000162, 0.005017, and 0.005206, respectively (Table 23). Genes related to jasmonic acid, salicylic acid, cycloheximide, brassinolide, ABA, ferric oxide, brassinosteroids, nitrogen, ethylene, and Gibberellin are enriched in V8.-LF. The P values are 8.91 E-05, 0.000607, 0.001392, 0.001604, 0.006216, 0.010594, 0.029935, 0.029935, 0.034, 0.037703, and 0.045091, respectively (Table 24). Genes related to sucrose, nitrogen, glucose, NO, jasmonic acid, methyl jasmonate, salicylic acid, ethylene, Ca++, and sodium chloride are enriched in V8-STK. The P values are 2.12E-05, 0.00188, 0.002404, 0.003041, 0.005565, 0.006988, 0.009356, 0.012852, 0.020218, and 0.046074, respectively (Table 25).
This application claims priority to and benefit of U.S. Provisional Patent Application Ser. No. 61/329,807, filed Apr. 30, 2010, the specification of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61329807 | Apr 2010 | US |
