WO2011159452A1 - Microrna polymorphisms conferring desirable phenotypes - Google Patents
Microrna polymorphisms conferring desirable phenotypes Download PDFInfo
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- WO2011159452A1 WO2011159452A1 PCT/US2011/038160 US2011038160W WO2011159452A1 WO 2011159452 A1 WO2011159452 A1 WO 2011159452A1 US 2011038160 W US2011038160 W US 2011038160W WO 2011159452 A1 WO2011159452 A1 WO 2011159452A1
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
- A01H6/4684—Zea mays [maize]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Definitions
- the field of the invention relates generally to plants with desirable phenotypic characteristics.
- the invention relates to identifying plant single nucleotide polymorphisms (SNPs) within microRNA regions that confer desirable agronomic phenotypes.
- SNPs plant single nucleotide polymorphisms
- the invention also relates to introgressing desirable agronomic phenotypes into plants by selecting plants comprising for one or more SNPs and breeding with such plants to confer such desirable agronomic phenotypes to plant progeny.
- a goal of plant breeding is to combine, in a single plant, various desirable traits.
- these traits can include greater yield and better agronomic quality.
- genetic loci that influence yield and agronomic quality are not always known, and even if known, their contributions to such traits are frequently unclear. Thus, new loci that can positively influence such desirable traits need to be identified and/or the abilities of known loci to do so need to be discovered.
- miRNAs are post-transcriptional regulators that bind to complementary sequences of target messenger RNA transcripts, and there is evidence that they play an important role in regulating gene activity. These 20-22 nucleotide noncoding RNAs have the ability to hybridize via base pairing with specific target mRNAs and downregulate the expression of these transcripts by mediating either RNA cleavage or translational repression.
- miRNAs have already been shown to play important roles in plant development, signal transduction, protein degradation, response to environmental stress and pathogen invasion, and regulate their own biogenesis (Zhang et al. (2006) Dev. Biol. 289:3-16). Further, miRNAs have been shown to control a variety of plant developmental processes including flowering time, leaf morphology, organ polarity, floral morphology, and root development (reviewed by Mallory and Vaucheret (2006) Nat. Genet. 38:S31-36).
- plant miRNAs share a high degree of complementarity with their targets (reviewed by Bonnet et al. (2006) New Phytol. 171 :451-468), and the predicted mRNA targets of plant miRNAs identified by computational methods encode a wide variety of proteins. Many of these proteins are transcription factors, which may have roles in development. Others are enzymes that have putative roles in mitochondrial metabolism, oxidative stress response, proteasome function, and lignification.
- miRNA families have been identified in Arabidopsis (reviewed by Meyers et al. (2006) Curr. Opin. Biotech. 17:1-8), and many of these miRNA sequences are associated with more than one locus, bringing the total number up to approximately 100.
- miRNAs identified by various investigators have not generally overlapped, it is assumed that the search for the entire set of miRNAs expressed by a given plant genome, the "miRNome,” is not yet complete. One reason for this might be that many miRNAs are expressed only under very specific conditions, and thus may have been missed by standard cloning efforts.
- miRNA regions i.e., a region of a chromosome coding for a mature miRNA, pre-miRNA and flanking sequences
- MAS marker-assisted selection
- MAB marker-assisted breeding
- MAS and MAB involves the use of one or more of the molecular markers for the identification and selection of those progeny plants that contain one or more loci that encode the desired traits. Such identification and selection may be based on selection of informative markers that are associated with desired traits.
- MAB can also be used to develop near-isogenic lines (NIL) harboring loci of interest, allowing a more detailed study of the effect each locus has on a desired trait, and is also an effective method for development of backcross inbred line (BIL) populations.
- NIL near-isogenic lines
- the present invention relates to methods of identifying a single nucleotide polymorphism associated with a plant trait.
- the single nucleotide polymorphism is located in a flanking sequence portion of a microRNA region.
- the single nucleotide polymorphism is located in a pre-miRNA portion of a microRNA region.
- the single nucleotide polymorphism is located in a mature miRNA portion of a microRNA region.
- the single nucleotide polymorphism is associated with miRNA169g, miRNA171 and miRNA393.
- the plant is maize.
- the plant trait is one or more of improved drought tolerance, improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
- the present invention also relates to methods of identifying a plant having an improved trait, where the trait is correlated with at least one single nucleotide polymorphism in a microRNA region of a plant genome.
- the single nucleotide polymorphism is located in a flanking sequence portion of a microRNA region.
- the single nucleotide polymorphism is located in a pre- miRNA portion of a microRNA region.
- the single nucleotide polymorphism is located in a mature miRNA portion of a microRNA region.
- the single nucleotide polymorphism is associated with miRNA169g, miRNA171 and miRNA393.
- the plant is maize.
- the plant trait is one or more of improved drought tolerance, improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
- the present invention also relates to isolated nucleic acids comprising a contiguous sequence of at least ten nucleotides selected from portions of the flanking sequence portion of miRNA169g, miRNA171 and miRNA393 microRNA regions that are associated with particular plant traits.
- the present invention also relates to methods of producing a transgenic plant having an improved trait and plants and plant parts produced thereby.
- Figures 1A-1 P Alignment of miRNA 169g sequence to identify SNPs.
- 169g mature miRNA and pre-miRNA are indicated by the identifiers mature_miRNA./123 (SEQ ID NO:43) and pre_miRNA71141 (SEQ ID NO:44), respectively.
- the wild type B73 sequence is indicated by the identifier, PUGHP42.R (SEQ ID NO:45).
- the miR169g locus has been mapped to the survey sequence, PUGHP42.R.
- FIGS. 2A-2L Alignment of miRNA 171a sequences to identify SNPs.
- the 171a mature miRNA and pre-miRNA are indicated by the identifiers mature_miR171a (SEQ ID NO:67) and zma-MIR171a (SEQ ID NO:68), respectively.
- the wild type B73 sequence is indicated by the identifier, chr4_240118217...240118861 (SEQ ID NO:69).
- the other corn lines aligned are: IJ6208./1643 (SEQ ID NO.70); A0100871626 (SEQ ID NO:71); BB3004./1644 (SEQ ID NO:72); CE8415./1573 (SEQ ID NO:73); DC4015./1587 (SEQ ID NO:74); FF609672619 (SEQ ID NO:75); PJ7065./1595 (SEQ ID NO:76); WR0588./1570 (SEQ ID NO:77); XF71 1071464 (SEQ ID NO:78); XO574471604 (SEQ ID NO:79); XPCC00371613 (SEQ ID NO:80); and XPFF003./1622 (SEQ ID NO:81).
- FIGS. 3A-3N Alignment of miRNA 393a sequences to identify SNPs.
- the mature miRNA and pre-miRNA are indicated by the identifiers mature_miRNA./123 (SEQ ID NO:82) and pre_miRNA./1127 (SEQ ID NO:83), respectively.
- the wild type B73 sequence is indicated by the identifier, chr2_736214...736992 (SEQ ID NO:84).
- the other corn lines aligned are: AO1008./1792 (SEQ ID NO:85); XF7110./1766 (SEQ ID NO:86); FF6096./1757 (SEQ ID NO:87); X05744./1755 (SEQ ID NO:88); ID5829./1612 (SEQ ID NO:89); FSNU505./1739 (SEQ ID NO:90); HT7049HL./1566 (SEQ ID NO:91); AX5707./1763 (SEQ ID NO:92); CC7752./1698 (SEQ ID NO:93); AF4031./1757 (SEQ ID NO:94); PJ7065./1782 (SEQ ID NO:95); HH5982./1566 (SEQ ID NO:96); CE8415./1733 (SEQ ID NO:97); IQ1332./1762 (SEQ ID NO:98); ID261871625 (SEQ ID NO:99); XPFF003./1746 (SEQ ID NO:100
- Figure 4 Procedure for phenotypic data analysis for the hybrid panel.
- phenotypic data analysis There were two purposes for phenotypic data analysis: data quality control and phenotypic adjustment for fitting association statistical models. Note that prior to phenotypic adjustment, there was also a data splitting process to subset the data according to various experimental conditions (e.g. locations, LD panels, and water treatments). The analysis for the inbred panel was similar but much simpler, because there were fewer data splits.
- Figure 5 shows the 169g amplicon (SEQ ID NO: 109).
- the SNPs are denoted with boxes.
- the pre-miRNA sequence is underlined, and the mature miRNA sequence is underlined and shaded.
- Figure 6 shows the 171 amplicon (SEQ ID NO:110).
- the SNPs are denoted with boxes.
- the pre-miRNA sequence is underlined, and the mature miRNA sequence is underlined and shaded.
- Figure 7 shows the 373 amplicon (SEQ ID ⁇ . 11).
- the SNPs are denoted with boxes.
- the pre-miRNA sequence is underlined, and the mature miRNA sequence is underlined and shaded.
- nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively.
- Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range.
- Amino acids may be referred to herein by either commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission.
- Nucleotides likewise, may be referred to by their commonly accepted single-letter codes. The terms described below are more fully explained by reference to the specification as a whole. It is further noted that, as used in this specification, the singular forms "a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent.
- Plant includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same.
- Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
- the term plant is also used in its broadest sense, including, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonas reinhardtii).
- Non-limiting examples of plants include plants from the genus Arabidopsis or the genus Oryza.
- plants include, but are not limited to, wheat, cauliflower, tomato, tobacco, corn, petunia, trees, etc.
- cereal crop is used in its broadest sense. The term includes, but is not limited to, any species of grass, or grain plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum, triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes or soybeans, etc.).
- crop or “crop plant” is used in its broadest sense. The term includes, but is not limited to, any species of plant or algae edible by humans or used as a feed for animals or used, or consumed by humans, or any plant or algae used in industry or commerce.
- plant part includes differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue).
- the plant tissue may be in plant or in a plant organ, tissue or cell culture.
- the aforementioned term also includes plant products, such as grain, fruits, and nuts.
- plant organ refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.
- genomic refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) present in each cell of an organism, or virus or organelle; (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.
- Progeny comprises any subsequent generation of a plant. Progeny will inherit, and stably segregate, genes and transgenes from its parent plant(s).
- a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature.
- a chimeric 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 found in nature.
- Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
- a plasmid vector can be used.
- the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention.
- the skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78- 86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern.
- Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.
- This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides.
- the construct may be transcribed to form an RNA, wherein the RNA may be capable of forming a double stranded RNA and/or hairpin structure.
- This construct may be expressed in the cell, isolated, or synthetically produced.
- the construct may further comprise a promoter, or other sequences that facilitate manipulation or expression of the construct.
- suppression As used herein, the terms “suppression”, “silencing” or “inhibition” are used interchangeably to denote the down-regulation of the expression of a product of a target sequence relative to its normal expression level in a wild type organism. Suppression includes expression that is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the wild type expression level.
- encodes or “encoding” refers to a DNA sequence that can be processed to generate an RNA and/or polypeptide.
- expression refers to production of a functional product, such as, the generation of an RNA transcript from an introduced construct, an endogenous DNA sequence, or a stably incorporated heterologous DNA sequence.
- the term may also refer to a polypeptide produced from an mRNA generated from any of the above DNA precursors.
- expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide).
- heterologous with respect to a 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.
- a nucleic acid it can be a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is modified from its native form in composition and/or genomic locus by deliberate human intervention.
- heterologous includes single nucleotide polymorphisms that may be introduced into a host organism.
- host cell refers to a cell that contains or into which is introduced a nucleic acid construct and supports the replication and/or expression of the construct.
- Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as fungi, yeast, insect, amphibian, nematode, or mammalian cells.
- the host cells are monocotyledonous or dicotyledonous plant cells.
- An example of a monocotyledonous host cell is a maize host cell.
- introduced means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing.
- nucleic acid fragment in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/ expression 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 nucleic acid fragment e.g., a recombinant DNA construct/ expression construct
- transduction 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
- gene as it applies to a 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.
- isolated refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
- microRNA or “miRNA” refers to an oligoribonucleic acid, which base pairs to a polynucleotide comprising the target sequence causing post- transcriptional regulation by transcript degredation or translational suppression.
- a “mature miRNA” refers to the miRNA generated from the processing of a “precursor miRNA” or “pre-miRNA”, which is the transcription product from a miRNA template.
- a “miRNA template” is an oligonucleotide region, or regions, in a nucleic acid construct that encodes the miRNA. The miRNA template may form a double-stranded polynucleotide, including a hairpin structure.
- domain refers to nucleic acid sequence(s) that are capable of eliciting a biological response in plants.
- the present invention concerns miRNAs comprised of at least 21 nucleotide sequences acting individually or in concert with other miRNA sequences; therefore a domain could refer to either individual miRNAs or groups of miRNAs. miRNA sequences associated with their backbone sequences could be considered domains useful for processing the miRNA into its active form.
- subdomains or “functional subdomains” refer to subsequences of domains that are capable of eliciting a biological response in plants. A miRNA could be considered a subdomain of a backbone sequence.
- Contiguous sequences or domains refer to sequences that are sequentially linked without added nucleotides intervening between the domains.
- Target sequence is used to mean the nucleic acid sequence that is selected for alteration (e.g., suppression) of expression, and is not limited to polynucleotides encoding polypeptides.
- the target sequence comprises a sequence that is substantially or fully complementary to the miRNA.
- the target sequence includes, but is not limited to, RNA, DNA, or a polynucleotide comprising the target sequence. As discussed in Bartel and Bartel ((2003) Plant Phys. 132:709-719), most microRNA sequences are 20 to 22 nucleotides with anywhere from 0 to 3 mismatches when compared to their target sequences.
- microRNA sequences such as the 21 nucleotide sequences of the present invention, may still be functional as shorter (20 nucleotide) or longer (22 nucleotide) sequences.
- some nucleotide substitutions particularly at the last two nucleotides of the 3' end of the microRNA sequence, may be useful in retaining at least some microRNA function.
- miR171a refers to the respective microRNAs from Zea mays and also encompass homologous and orthologous microRNAs in other plants.
- Homologous microRNAs include those with 70% or greater sequence homology to the above-noted miRNAs in Zea mays, for example, at least about 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%. Homologous and orthologous microRNAs will also share a similar chromosomal location.
- single nucleotide polymorphism refers to a single nucleotide variation in a gene or contiguous region upstream or downstream from a gene that differs from the typical genomic sequence for that organism.
- a “miRNA region” refers to sequences upstream, downstream, or within a miRNA template that contribute to folding or processing of the miRNA transcript or regulating transcription of the miRNA, i.e., features of the levels, spatial distribution, and/or temporal profile of the miRNA expression. Such miRNA regions can be identified, for example, based upon the presence of at least one single nucleotide polymorphism (SNP) or mutation that enhances or decreases transcript level of a mature miRNA.
- SNP single nucleotide polymorphism
- nucleic acid means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms “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 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 (Cor T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
- nucleic acid library is used to refer to a collection of isolated DNA or RNA molecules that comprise and substantially represent the entire transcribed fraction of a genome of a specified organism or of a tissue from that organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references (see, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning— A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
- operably linked includes reference to a functional linkage of at least two sequences. Operably linked includes linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
- polypeptide means proteins, protein fragments, modified proteins, amino acid sequences and synthetic amino acid sequences.
- the polypeptide can be glycosylated or not.
- promoter refers to a nucleic acid fragment, e.g., a region of
- this nucleic acid fragment is capable of controlling transcription of another nucleic acid fragment.
- sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
- Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
- stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
- stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides).
- Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
- Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1 % SDS at 37 °C, and a wash in 0.5x to 1x SSC at 55 to 60 °C
- Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at 37 °C, and a wash in 0.1 x SSC at 60 to 65 °C.
- the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. 7 " m is reduced by about 1 °C for each 1 % of mismatching; thus, 7 " m hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10 °C Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (7 m ) for the specific sequence and its complement at a defined ionic strength and pH.
- T m of less than 45 °C (aqueous solution) or 32 °C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used.
- An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley- Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120, or 240 minutes.
- reliable detection and “reliably detected” are defined herein to mean the reproducible detection of measurable, sequence-specific signal intensity above background noise.
- transgenic refers to a plant or a cell that comprises within its genome a heterologous polynucleotide.
- the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on, or heritable, to successive generations.
- the heterologous polynucleotide may be integrated into the genome alone or as part of an expression construct.
- Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
- transgenic does not encompass the alteration of the genome (chromosomal or extrachromosomal) 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.
- vector refers to a small nucleic acid molecule (plasmid, virus, bacteriophage, artificial or cut DNA molecule) that can be used to deliver a polynucleotide of the invention into a host cell. Vectors are capable of being replicated and contain cloning sites for introduction of a foreign polynucleotide. Thus, expression vectors permit transcription of a nucleic acid inserted therein.
- Polynucleotide sequences may have substantial identity, substantial homology, or substantial complementarity to the selected region of the target gene.
- substantially identity and “substantial homology” indicate sequences that have sequence identity or homology to each other.
- sequences that are substantially identical or substantially homologous will have about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity wherein the percent sequence identity is based on the entire sequence and is determined by GAP alignment using default parameters (GCG, GAP version 10, Accelrys, San Diego, Calif.).
- GAP uses the algorithm of Needleman and Wunsch (Mol. Biol.
- sequences which have 100% identity are identical.
- Substantial complementarity refers to sequences that are complementary to each other, and are able to base pair with each other. In describing complementary sequences, if all the nucleotides in the first sequence will base pair to the second sequence, these sequences are fully or completely complementary.
- 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.
- PTGS post-transcriptional gene silencing
- 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).
- Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA.
- dsRNAs double-stranded RNAs
- the presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.
- dsRNAs short interfering RNAs
- dicer a ribonuclease III enzyme referred to as "dicer."
- Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., Nature 409:363 2001) and/or pre miRNAs into miRNAs.
- short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al., Genes Dev. 15:188 2001).
- Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001 , Science 293:834).
- the RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., Genes Dev.
- RISC RNA-induced silencing complex
- RNA interference can also involve small RNA (e.g., microRNA, or miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences ⁇ see, e.g., Allshire, Science 297:1818-1819 2002; Volpe et al., Science 297:1833-1837 2002; Jenuwein, Science 297:2215- 2218 2002; and Hall et al., Science 297:2232-2237 2002).
- miRNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post- transcriptional level.
- 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 function, at least in part, by base-pairing to complementary RNA or
- RNA 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 are noncoding RNAs of about 19 to about 24 nucleotides
- miRNA hairpin precursors originate as longer polyadenylated transcripts, and several different miRNAs and associated hairpins can be present in a single transcript (Lagos-Quintana et al (2001) Science 294:853-858; Lee et al., (2002) EMBO J. 21 :4663-4670).
- the target sites are located in the 3' UTRs of the target mRNAs (Lee et al (1993) Cell 75:843-854; Wightman et al (1993) Cell 75:855-862; Reinhart et al (2000) Nature 403:901-906; Slack et al., Mol. Cell. 5:659-669 2000), and there are several mismatches between the lin-4 and let-7 miRNAs and their target sites.
- lin-4 or let-7 miRNA may downregulate steady-state levels of the protein encoded by the target mRNA without affecting the transcript itself (Olsen and Ambros, Dev. Biol. 216:671-680 1999).
- miRNAs appear to cause specific RNA cleavage of the target transcript within the target site, and that this cleavage step requires 100% complementarity between the miRNA and the target transcript (Hutvagner and Zamore, (2002) Science 297:2056-2060; Llave et al., Plant Cell 14:1605-1619 2002).
- miRNAs may contribute to at least two pathways of target gene regulation: Protein downregulation when target complementarity is ⁇ 100%, and RNA cleavage when target complementarity is 100%.
- MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nucleotide short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants (Hamilton and Baulcombe 1999; Hammond et al., 2000; Zamore et al., 2000; Elbashir et al., 2001 ), and are incorporated into an RNA- induced silencing complex (RISC) that is similar or identical to that seen for RNAi.
- siRNAs 21-25 nucleotide short interfering RNAs
- PTGS posttranscriptional gene silencing
- An aspect of the present invention is a method for identifying single nucleotide polymorphisms in miRNA regions using association mapping.
- Association mapping including genome-wide association mapping and candidate-gene association mapping, has emerged as a tool to resolve complex trait variation down to the sequence level.
- Genome-wide association mapping is conducted to find signals of association for various complex traits by surveying genetic variation in the whole genome.
- Candidate- gene association mapping relates polymorphisms in selected candidate genes that could control phenotypic variation for specific traits.
- Association mapping relies on chromosomal recombination opportunities over a large number of generations, in the history of a species, which allows the removal of association between a QTL and any marker not tightly linked to it, thus improving the rate of discovery of true association (Jannink and Walsh, Quantitative Genetics, Genomics and Plant Breeding, Kang, Ed. CAB International, (2002) pp. 59-68).
- MTA marker-trait association
- LD linkage disequilibrium
- LD mapping relies on linkage disequilibrium, which is defined as the non-random association of alleles from two different loci (genes or markers) in a natural population.
- LD mapping assumes that the main cause for LD is linkage that binds loci on the same chromosome together in transmission to next generation.
- linkage disequilibrium which is defined as the non-random association of alleles from two different loci (genes or markers) in a natural population.
- linkage disequilibrium is defined as the non-random association of alleles from two different loci (genes or markers) in a natural population.
- linkage disequilibrium is defined as the non-random association of alleles from two different loci (genes or markers) in a natural population.
- LD mapping assumes that the main cause for LD is linkage that binds loci on the same chromosome together in transmission to next generation.
- each chromosome has been shuffled
- LD mapping identifies genes of interest through genetic markers on the LD blocks where the genes are located. This is done by detecting significant associations between the markers and the traits that the genes affect with a sample of unrelated individuals or a sample of unrelated pedigrees that are genotyped on a selected set of markers covering candidate gene regions or the whole genome, and phenotyped on a set of traits of interest.
- LD mapping Compared with traditional linkage mapping methods that are typically based on artificial biparental segregating populations (e.g., F2, BC, DH, RIL, etc.), LD mapping generally produces better mapping resolution, because of the smaller sizes of LD blocks. In addition, LD mapping is useful in identifying more than two functional alleles at associated markers in a germplasm. Further, LD mapping is efficient for evaluating natural populations.
- Linkage disequilibrium may be caused by factors other than linkage, such as mutation, migration, inbreeding, and genetic drift, inter alia. Consequently, LD mapping can be prone to false positives or spurious MTAs. Spurious MTAs are marker-trait associations between unlinked or distantly linked loci. Another consideration is the sample population structure. Population structure has been has been studied extensively, and effective statistical approaches have been developed to significantly reduce false positives in human genetics and in plants as well (Yu et al. (2006) Nat. Genet. 38:203-208). In addition, LD mapping requires high-density marker coverage on the genome in order to capture as many tiny LD blocks as possible. This issue has been largely overcome by high-throughput genotyping technology. However, other considerations in experimental design include precision and accuracy of phenotype acquisition in addition to throughput (Myles et al. (2009) Plant Cell 21 :2194-2202).
- Markers selected for association mapping are often chosen randomly with the goal of having the greatest number of markers spaced evenly across the genome.
- Another strategy known as candidate gene strategy, is to make markers to score the alleles of genes that are suspected to influence the phenotype that one will evaluate.
- the present application discloses a third strategy (i.e., using markers to distinguish alleles of miRNAs that are associated with trait of interest). This third strategy has the advantage that miRNAs regulate many genes, and the genes they regulate often regulate many other genes.
- Another aspect of the invention is methods for suppressing a target sequence.
- the methods employ any constructs in which a miRNA is designed to identify a region of the target sequence, and inserted into the construct.
- the miRNA is provided in a nucleic acid construct which, when transcribed into RNA, is predicted to form a hairpin structure which is processed by the cell to generate the miRNA, which then suppresses expression of the target sequence.
- the miRNA produced suppresses expression of the targeted sequence.
- the target sequence can be an endogenous plant sequence, or a heterologous transgene in the plant.
- the invention includes constructs comprising one or more of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
- the methods provided can be practiced in any organism in which a method of transformation is available, and for which there is at least some sequence information for the target sequence, or for a region flanking the target sequence of interest. It is also understood that two or more sequences could be targeted by sequential transformation, co-transformation with more than one targeting vector, or the construction of a DNA construct comprising more than one miRNA sequence.
- the methods of the invention may also be implemented by a combinatorial nucleic acid library construction in order to generate a library of miRNAs directed to random target sequences.
- the library of miRNAs could be used for high-throughput screening for gene function validation.
- sequences of interest include, for example, those genes involved in regulation or information, such as zinc fingers, transcription factors, homeotic genes, or cell cycle and cell death modulators, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins.
- Other categories of target sequences include genes affecting agronomic traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest also included those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting, for example, kernel size, sucrose loading, and the like. The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality, and quantity of essential amino acids, and levels of cellulose.
- genes of the phytic acid biosynthetic pathway could be suppressed to generate a high available phosphorous phenotype.
- phytic acid biosynthetic enzymes including inositol polyphosphate kinase-2 polynucleotides, disclosed in PCT International Publication No. WO 02/059324, inositol 1 ,3,4- trisphosphate 5/6-kinase polynucleotides, disclosed in PCT International Publication No. WO 03/027243, and myo-inositol 1 -phosphate synthase and other phytate biosynthetic polynucleotides, disclosed in PCT International Publication No.
- WO 99/05298 Genes in the lignification pathway could be suppressed to enhance digestibility or energy availability. Genes affecting cell cycle or cell death could be suppressed to affect growth or stress response. Genes affecting DNA repair and/or recombination could be suppressed to increase genetic variability. Genes affecting flowering time could be suppressed, as well as genes affecting fertility. Any target sequence could be suppressed in order to evaluate or confirm its role in a particular trait or phenotype, or to dissect a molecular, regulatory, biochemical, or proteomic pathway or network.
- Target sequences further include coding regions and non-coding regions such as promoters, enhancers, terminators, introns and the like, which may be modified in order to alter the expression of a gene of interest.
- an intron sequence can be added to the 5' region to increase the amount of mature message that accumulates (see, e.g., Buchman and Berg, (1988) Mol. Cell. Biol. 8:4395-4405; and Callis et al (1987) Genes Dev. 1 :1 83-1200).
- the target sequence may be an endogenous sequence, or may be an introduced heterologous sequence, or transgene.
- the methods may be used to alter the regulation or expression of a transgene, or to remove a transgene or other introduced sequence such as an introduced site-specific recombination site.
- the target sequence may also be a sequence from a pathogen, for example, the target sequence may be from a plant pathogen such as a virus, a mold or fungus, an insect, or a nematode.
- a miRNA could be expressed in a plant that, upon infection or infestation, would target the pathogen and confer some degree of resistance to the plant.
- promoters can be used, these promoters can be selected based on the desired outcome. It is recognized that different applications will be enhanced by the use of different promoters in plant expression cassettes to modulate the timing, location and/or level of expression of the miRNA.
- plant expression cassettes may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
- constitutive, tissue-preferred or inducible promoters can be employed.
- constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1 '- or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenate promoter (U.S. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter and other transcription initiation regions from various plant genes known to those of skill. If low level expression is desired, weak promoter(s) may be used.
- Weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (PCT International Publication No. WO 99/43838 and U.S. Patent No. 6,072,050), the core 35S CaMV promoter, and the like.
- Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Patent No. 6,177,611.
- inducible promoters examples include the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the ln2-2 promoter which is safener induced (U.S. Patent No. 5,364,780), the ERE promoter which is estrogen induced, and the Axigl promoter which is auxin induced and tapetum specific but also active in callus (PCT US01/22169).
- promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
- An exemplary promoter is the anther specific promoter 5126 (U.S. Patent Nos. 5,689,049 and 5,689,051).
- seed preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A. et al. (1986) Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21 ):6426; and Kloesgen, R. B. et al. (1986) Mol. Gen. Genet. 203:237-244. Promoters that express in the embryo, pericarp, and endosperm are disclosed in U.S. Patent No. 6,225,529 and PCT International Publication No. WO 00/12733.
- an inducible promoter particularly from a pathogen-inducible promoter.
- promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1 ,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:1 11-116. See also PCT International Publication No. WO 99/43819.
- promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331 ; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427- 2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al.
- a wound-inducible promoter may be used in the constructions of the polynucleotides.
- Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotech. 14:494-498); wunl and wun2, U.S. Patent No. 5,428,148; winl and wing (Stanford et al. (1989) Mol. Gen. Genet. 215:200- 208); systemin (McGurl et al.
- Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
- the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
- Chemical inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid.
- promoters of interest include steroid steroid-responsive promoters (see, for example, the glucocorticoid- inducible promoter in Schena et al. (1991) Proc. ⁇ Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline- repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156.
- Tissue-preferred promoters can be utilized to target enhanced expression of a sequence of interest within a particular plant tissue.
- Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7)792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 1 12(3):1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.
- Leaf-preferred promoters are known in the art. See, e.g., Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
- the promoters of cab and rubisco can also be used. See, e.g., Simpson et al. (1958) EMBO J. 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.
- Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051 -1061 (root specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.
- MAS mannopine synthase
- TRV gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics.
- Additional root- preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4) :681-691. See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459, 252; 5,401 ,836; 5,110,732; and 5,023,179.
- the phaseolin gene (Murai et al. (1983) Science 23:476-482 and Sengopta-Gopalen et al. (1988) PNAS 82:3320-3324.
- Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing the DNA construct include microinjection (Crossway et al. (1986) Biotechniques 4:320-334; and U.S. Patent No. 6,300,543), sexual crossing, electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-med ated transformation (Townsend et al., U.S. Patent No. 5,563,055; and U.S. Patent No.
- the nucleotide constructs may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that useful promoters encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, e.g., U.S. Patent Nos. 5,889,191 , 5,889,190, 5,866,785, 5,589,367 and 5,316,931.
- DNA constructs containing miRNA genes and their corresponding upstream and downstream regulatory regions may be integrated of the into the host cell chromosome according to conventional methods, e.g., by homologous recombination or other methods of integration, including targeted integration at a particular host chromosomal site.
- transient expression may be desired.
- standard transient transformation techniques may be used. Such methods include, but are not limited to viral transformation methods, and microinjection of DNA or RNA, as well other methods well known in the art.
- the cells from the plants that have stably incorporated the nucleotide sequence may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic imparted by the nucleotide sequence of interest and/or the genetic markers contained within the target site or transfer cassette. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
- Initial identification and selection of cells and/or plants comprising the DNA constructs may be facilitated by the use of marker genes.
- Gene targeting can be performed without selection if there is a sensitive method for identifying recombinants, for example if the targeted gene modification can be easily detected by PCR analysis, or if it results in a certain phenotype. However, in most cases, identification of gene targeting events will be facilitated by the use of markers.
- Useful markers include positive and negative selectable markers as well as markers that facilitate screening, such as visual markers.
- Selectable markers include genes carrying resistance to an antibiotic such as spectinomycin (e.g. the aada gene, Svab et al. 1990 Plant Mol. Biol.
- streptomycin e.g., aada, or SPT, Svab et al. 1990 Plant Mol. Biol. 14:197; Jones et al. 1987 Mol. Gen. Genet. 210:86
- kanamycin e.g., nptll, Fraley et al. 1983 PNAS 80:4803
- hygromycin e.g., HPT, Vanden Elzen et al. 1985 Plant Mol. Biol. 5:299
- gentamycin Hayford et al. 1988 Plant Physiol. 86:1216)
- phleomycin zeocin
- bleomycin Hille et al. (1986) Plant Mol. Biol.
- Negative selectable markers include cytosine deaminase (codA) (Stougaard (1993) Plant J. 3:755-761 ), tms2 (DePicker et al. (1988) Plant Cell Rep.
- nitrate reductase Nussame et al. (1991) Plant J. 1 :267-274
- SU1 O'Keefe et al. (1994) Plant Physiol. 105:473-482
- aux-2 from the Ti plasmid of Agrobacterium
- thymidine kinase Screenable markers include fluorescent proteins such as green fluorescent protein (GFP) (Chalfie et al. (1994) Science 263:802; U.S. Patent No. 6,146,826; U.S. Patent No. 5,491 ,084; and PCT International Publication No.
- GFP green fluorescent protein
- reporter enzymes such as 13-glucuronidase (GUS) (Jefferson R. A. (1987) Plant Mol. Biol. Rep. 5:387; U.S. Patent No. 5,599,670; and U.S. Patent No. 5,432,081), 13-galactosidase (lacZ), alkaline phosphatase (AP), glutathione S- transferase (GST) and luciferase (U.S. Patent No. 5,674,713; and Ow et al. (1986) Science 234(4778):856-859), visual markers like anthocyanins such as CRC (Ludwig et al.
- R gene family e.g., Lc, P, S
- One or more markers may be used in order to select and screen for gene targeting events.
- One common strategy for gene disruption involves using a target modifying polynucleotide in which the target is disrupted by a promoterless selectable marker. Since the selectable marker lacks a promoter, random integration events generally do not lead to transcription of the gene. Gene targeting events will put the selectable marker under control of the promoter for the target gene. Gene targeting events are identified by selection for expression of the selectable marker.
- Another common strategy utilizes a positive-negative selection scheme. This scheme utilizes two selectable markers, one that confers resistance (R+) coupled with one that confers sensitivity (S+), each with a promoter.
- Another aspect of the invention concerns a plant, cell, and seed comprising the construct and/or the miRNA.
- the cell will be a cell from a plant, but other prokaryotic or eukaryotic cells are also contemplated, including but not limited to viral, bacterial, yeast, insect, nematode, or animal cells.
- Plant cells include cells from monocots and dicots.
- the invention also provides plants and seeds comprising the construct and/or the miRNA.
- Genomic DNA amplicons containing the miR169g, miR171a, and miR393 regions and the upstream and downstream flanking sequences were amplified using the primers shown in Table 1 from a Maize genomic DNA library derived from a diverse panel of inbred lines. SNPs were identified by aligning the sequences from the Maize lines using SeqScape Software Version 2.5 from Applied Biosystems ( Figures 1A-1 P, 2A-2L, and 3A-3N).
- a TAQMAN® genotyping assay (Applied Biosystems) was developed to evaluate the prevalence of SNPs in the three miRNAs, miR171 and miR393 regions on approximately 700 base pair amplicons (Livak et al. (1995) Nat. Genetics 9:341-342).
- allelic discrimination assays a PCR assay includes a forward and reverse primer and a specific, fluorescent, dye-labeled probe for each of two alleles. The probes contain different fluorescent reporter dyes (VIC® and FAM, or TET and FAM) to differentiate the amplification of each allele.
- FAM is 6-carboxyfluoroscein
- TET is 6-carboxy-4,7,2',7'- tetrachlorofluorescein
- VIC® is a proprietary dye (Applied Biosystems).
- a non- fluorescent quencher on each probe suppresses the fluorescence until amplification by PCR.
- each probe anneals specifically to complementary sequences between the forward and reverse primer sites.
- Taq DNA polymerase then cleaves the probes that are hybridized to each allele. Cleavage separates the reporter dye from the quencher, which results in increased fluorescence by the reporter dye.
- the fluorescent signals generated by PCR amplification indicate that one or both alleles are present in the sample.
- the probe In addition to the nonfluorescent quencher, the probe also contains a minor groove binder at the 3' end, which results in an increased melting temperature (7 " m ), thereby allowing high specificity with the use of shorter oligos. These probes therefore exhibit greater 7 m differences when hybridized to matched and mismatched templates, which provides more accurate allelic discrimination. Probes of this type can be manufactured at either ABI (MGBTM quencher) or Biosearch Technologies (BHQPLUSTM quencher). At the end of PCR thermal cycling, fluorescence of the two reporter dyes is measured on an ABI 7900 Sequence Detection System. An increase in fluorescence for one dye indicates homozygosity for the corresponding allele. Increase in both fluorescent signals indicates heterozygosity. Table 2: TAQMAN® Primers and Probes
- TAQMAN® allelic discrimination assays for association with drought tolerance
- plants were selected based on their known phenotypic status and compared to the genotype at the specific SNP location.
- DNA was extracted from leaf tissue of seedlings 7-10 days after planting.
- DNA can be extracted from plant tissue in a variety of ways, including the CTAB method, sodium hydroxide, and the Dellaporta method.
- DNA is diluted in TE buffer (10 mM Tris HCI, pH 7.5, 1 mM EDTA) and stored at 4°C until used in PCR reactions. PCR reactions were set up in 5 pL final volumes according to Table 3.
- thermocycle programs were run.
- the ABI 7900 Sequence Detection System was used to visualize the results of an allelic discrimination SNP assay.
- SDS Sequence Detection System
- allele calls were determined based on the fluorescence for the two dyes measured in each sample.
- Table 5 shows the SNP positions and allele types for amplicons 169g, 393, and 171a.
- Table 6 is the summary of haplotypes observed in plants and the number of occurrences.
- An association mapping study begins with development of a population sample, continues with genotyping and phenotyping all individuals in the sample, and ends with data analysis and result summary.
- the population sample is a set of unrelated individuals (with no known pedigree relationships), which is called the linkage disequilibrium (LD) panel, or a set of unrelated pedigrees (Cardon and Bell (2001) Nat. Rev. Genet. 2:91-99).
- An association study needs to make many strategic decisions around the population sample, genetic markers, genotyping platform, experimental design (e.g. treatments, locations and repetitions) for phenotyping with field trials, and the choice of appropriate statistical procedure and methods.
- MTA results from the study depend heavily on the size and composition of the population sample, genomic coverage of genetic markers (candidate-genes based or genome-wide), precision of genotyping and phenotyping, and appropriate use of statistical procedure and methods.
- the population samples used in this study were from two commercially establish LD panels of diverse inbred lines, an inbred maize panel and a hybrid maize panel.
- the hybrid panel further consisted of two subpanels: the non-stiff stalk (NSS) panel and the stiff stalk (SS) panel, while the inbred panel is a mixture of both SS and NSS inbreds.
- NSS and SS are the two main targeted heterotic groups in maize.
- the inbred panel and both hybrid subpanels each consisted of approximately 600 inbred lines selected from a platform of 2,075 inbreds that represent the wide genetic diversity and maturity groups (early, intermediate, and late) in the maize germplasm.
- the inbred panel was genotyped and phenotyped directly using the inbred panel lines.
- the hybrid panel was genotyped on the inbred panel as well, and phenotyping was conducted on the hybrids of the inbred panel with a commercially important inbred as the tester.
- the combination of phenotypic data on both inbreds and hybrids was intended to study the effects of genetic backgrounds (homozygous and heterozygous) on MTAs.
- the two LD panels were each phenotyped in one year at multiple locations. Two water treatments were assessed; normal irrigation (WET) and flowering-time drought stress (DRY) were conducted with both panels. These experiments assessed the effects of MTAs on yield and drought tolerance under different irrigation conditions.
- each subpanel of the hybrid maize panel was grown at 5 locations with WET treatment, and 3 locations with DRY treatment. Three repetitions were applied for WET treatment, and 6 repetitions for DRY treatment, at all locations where the treatment was applied.
- phenotypic data analysis There were two purposes for phenotypic data analysis: data quality control (QC) and phenotypic adjustment for fitting association statistical models.
- the procedure for analyzing the phenotypic data on the hybrid panel is shown in the flowchart in Figure 4.
- the phenotypic data were split, according to various experimental conditions, in order to detect MTAs that might be caused by various types of gene by environment interactions. 938 lines (434 NSS, 504 SS) were phenotyped for 13 trait in DRY and WET conditions. Data splitting was carried out prior to phenotypic adjustment for model fitting. It was intended to subset the cleaned data according to various experimental conditions including water treatments. Data for each split was then analyzed separately to detect MTAs under particular experimental conditions to capture effects from G*E and GxG interactions.
- split-specific phenotypic adjustment was done to remove all non-genetic effects (or design-of-experiment (DOE) effects), including effects from locations, repetitions, LD panels, water treatments, etc., depending on the data split in question.
- DOE design-of-experiment
- Phenotypic data adjustment is a necessary step for fitting the GLM / MLM association models.
- MLM which relies on a few statistical assumptions, including independency between fitted values and random residuals, and normal distribution for random residuals. Violation of these assumptions would affect the reliability and accuracy of the final MTA results (p values, etc.). Therefore, it was important to determine the quality (model fitness) of the adjusted phenotypic data, so that the MTA results from the adjusted data would not be over-interpreted.
- Grain moisture traits GMSAP and GMSTP
- grain yield traits YGSMN, YGSAN, YGSMN/GMSTP, and YGSAN/GMSAP
- Grain moisture traits GMSAP and GMSTP
- grain yield traits YGSMN, YGSAN, YGSMN/GMSTP, and YGSAN/GMSAP
- YGHMN and YGHAN two yield traits unadjusted for standard moisture
- Morphological traits ERHTN and PLHTN
- flowering time traits SK5N, ASIDN, and POL5N
- the SNP at position 701 of the 169g amplicon i.e., marker SM1480AQ
- GMSAP grain moisture adjusted percentage
- Table 8 is similar to Table 7, but cross-references Table 6 and shows the effect of haplotype on a particular plant trait. For example, looking at row 23, one sees that marker SM1480 is associated with grain moisture adjusted percentage (GMSAP), consistent with the first row of Table 7 discussed above. As shown in Table 7, four combinations of alleles (out of eight possible) in the SM1480 marker are present in the 1064 plants examined.
- GMSAP grain moisture adjusted percentage
- the most frequent haplotype resulting in this favorable phenotype is the "C” haplotype (i.e., a "T” at position 174 of the 169g amplicon, a "C” at position 259 of the 169g amplicon, and a “T” at position 701 of the 169g amplicon), while the most frequent haplotype resulting in an unfavorable phenotype is the "A” haplotype (i.e., a "A” at position 174 of the 169g amplicon, a "C” at position 259 of the 169g amplicon, and a "C” at position 701 of the 169g amplicon).
- the effect of the "C” haplotype on grain moisture adjusted percentage ranges from 0.84279 to 1.5428 and the mean is 1.18, meaning that plants having these variant alleles have 1.18% less moisture at harvest (which is desirable for the reasons described above).
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Abstract
Methods of identifying a single nucleotide polymorphism associated with a plant trait and methods of identifying a plant having an improved trait. The plant trait is correlated with at least one single nucleotide polymorphism in a microRNA region of a plant genome. Isolated nucleic acids, transgenic plants, and methods of producing the same are also disclosed.
Description
MICRORNA POLYMORPHISMS CONFERRING DESIRABLE PHENOTYPES
FIELD OF THE INVENTION
The field of the invention relates generally to plants with desirable phenotypic characteristics. The invention relates to identifying plant single nucleotide polymorphisms (SNPs) within microRNA regions that confer desirable agronomic phenotypes. The invention also relates to introgressing desirable agronomic phenotypes into plants by selecting plants comprising for one or more SNPs and breeding with such plants to confer such desirable agronomic phenotypes to plant progeny.
BACKGROUND OF THE INVENTION
A goal of plant breeding is to combine, in a single plant, various desirable traits. For field crops such as corn, these traits can include greater yield and better agronomic quality. However, genetic loci that influence yield and agronomic quality are not always known, and even if known, their contributions to such traits are frequently unclear. Thus, new loci that can positively influence such desirable traits need to be identified and/or the abilities of known loci to do so need to be discovered.
Previous studies have focused primarily on the identification and manipulation of candidate genes that encode proteins, such as transcription factors. These genes could encode proteins that directly affect the physiology of the plant or transcription factors that regulate these effector genes.
miRNAs are post-transcriptional regulators that bind to complementary sequences of target messenger RNA transcripts, and there is evidence that they play an important role in regulating gene activity. These 20-22 nucleotide noncoding RNAs have the ability to hybridize via base pairing with specific target mRNAs and downregulate the expression of these transcripts by mediating either RNA cleavage or translational repression.
Numerous efforts are ongoing to discover miRNA genes that influence plant traits. These efforts rely on classic molecular biology cloning and expression techniques, as well as computational methods (see, e.g., U.S. Patent Application
Publication No. 200701 18918). miRNAs have already been shown to play important roles in plant development, signal transduction, protein degradation, response to environmental stress and pathogen invasion, and regulate their own biogenesis (Zhang et al. (2006) Dev. Biol. 289:3-16). Further, miRNAs have been shown to control a variety of plant developmental processes including flowering time, leaf morphology, organ polarity, floral morphology, and root development (reviewed by Mallory and Vaucheret (2006) Nat. Genet. 38:S31-36).
In general, plant miRNAs share a high degree of complementarity with their targets (reviewed by Bonnet et al. (2006) New Phytol. 171 :451-468), and the predicted mRNA targets of plant miRNAs identified by computational methods encode a wide variety of proteins. Many of these proteins are transcription factors, which may have roles in development. Others are enzymes that have putative roles in mitochondrial metabolism, oxidative stress response, proteasome function, and lignification.
At least 30 miRNA families have been identified in Arabidopsis (reviewed by Meyers et al. (2006) Curr. Opin. Biotech. 17:1-8), and many of these miRNA sequences are associated with more than one locus, bringing the total number up to approximately 100. As the particular miRNAs identified by various investigators have not generally overlapped, it is assumed that the search for the entire set of miRNAs expressed by a given plant genome, the "miRNome," is not yet complete. One reason for this might be that many miRNAs are expressed only under very specific conditions, and thus may have been missed by standard cloning efforts. A study by Sunkar and Zhu (2004, Plant Cell 1 (6):2001-2019) suggests that, indeed, miRNA discovery may be facilitated by choosing "non-standard" growth conditions for library construction. Sunkar and Zhu identified novel miRNAs in a library consisting of a variety of stress-induced tissues and they demonstrated induction of some of these miRNAs by drought, cold and other stresses, suggesting a role for miRNAs in stress responses. This conclusion is reinforced by the observation that an miRNA targeting genes in the sulfur assimilation pathway was shown to be induced under conditions of sulfate starvation (Jones- Rhoades and Bartel (2004) Mol. Cell. 14:787-799).
However, what has gone completely unappreciated up to this point is that polymorphisms present in miRNA regions (i.e., a region of a chromosome coding for a
mature miRNA, pre-miRNA and flanking sequences) have a measurable impact on plant phenotype. Accordingly, using this knowledge a skilled artisan can manipulate plants and plant materials using both and classic molecular biology techniques and traditional breeding techniques to introduce desirable traits into plant varieties. For example, desirable loci can be introgressed into commercially available plant varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB involves the use of one or more of the molecular markers for the identification and selection of those progeny plants that contain one or more loci that encode the desired traits. Such identification and selection may be based on selection of informative markers that are associated with desired traits. MAB can also be used to develop near-isogenic lines (NIL) harboring loci of interest, allowing a more detailed study of the effect each locus has on a desired trait, and is also an effective method for development of backcross inbred line (BIL) populations. BRIEF SUMMARY OF THE INVENTION
The present invention relates to methods of identifying a single nucleotide polymorphism associated with a plant trait. In some embodiments, the single nucleotide polymorphism is located in a flanking sequence portion of a microRNA region. In other embodiments, the single nucleotide polymorphism is located in a pre-miRNA portion of a microRNA region. In yet other embodiments, the single nucleotide polymorphism is located in a mature miRNA portion of a microRNA region. In still other embodiments, the single nucleotide polymorphism is associated with miRNA169g, miRNA171 and miRNA393.
In some embodiments, the plant is maize. In some embodiments the plant trait is one or more of improved drought tolerance, improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted
percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
The present invention also relates to methods of identifying a plant having an improved trait, where the trait is correlated with at least one single nucleotide polymorphism in a microRNA region of a plant genome. In some embodiments, the single nucleotide polymorphism is located in a flanking sequence portion of a microRNA region. In other embodiments, the single nucleotide polymorphism is located in a pre- miRNA portion of a microRNA region. In yet other embodiments, the single nucleotide polymorphism is located in a mature miRNA portion of a microRNA region. In still other embodiments, the single nucleotide polymorphism is associated with miRNA169g, miRNA171 and miRNA393.
In some embodiments, the plant is maize. In some embodiments the plant trait is one or more of improved drought tolerance, improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
The present invention also relates to isolated nucleic acids comprising a contiguous sequence of at least ten nucleotides selected from portions of the flanking sequence portion of miRNA169g, miRNA171 and miRNA393 microRNA regions that are associated with particular plant traits.
The present invention also relates to methods of producing a transgenic plant having an improved trait and plants and plant parts produced thereby.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention are better understood when the following Detailed Description is read with reference to the accompanying figures.
Figures 1A-1 P. Alignment of miRNA 169g sequence to identify SNPs. The
169g mature miRNA and pre-miRNA are indicated by the identifiers mature_miRNA./123 (SEQ ID NO:43) and pre_miRNA71141 (SEQ ID NO:44), respectively. The wild type B73 sequence is indicated by the identifier, PUGHP42.R (SEQ ID NO:45). The miR169g locus has been mapped to the survey sequence, PUGHP42.R. The other corn lines aligned are: ID7002./1775 (SEQ ID NO:46); AA394171769 (SEQ ID NO:47); AF403171743 (SEQ ID NO:48); AX5707./1782 (SEQ ID NO:49); BB3004./1775 (SEQ ID NO:50); CC803271763 (SEQ ID NO:51); CE841571747 (SEQ ID NO:52); FSNU505./1735 (SEQ ID NO:53); HT7049HL71754 (SEQ ID NO:54); ID2618./1738 (SEQ ID NO:55); ID5829./1759 (SEQ ID NO:56); IJ6208./1719 (SEQ ID NO:57); IQ1332./1775 (SEQ ID NO:58); WR058871759 (SEQ ID NO:59); XF71 1071788 (SEQ ID NO:60); X05744./1759 (SEQ ID NO:61); XPFF003./1771 (SEQ ID NO:62); XPCC003./1731 (SEQ ID NO:63); PJ7065./1732 (SEQ ID NO:64); FF6096./1784 (SEQ ID NO:65); and CC775271770 (SEQ ID NO:66).
Figures 2A-2L. Alignment of miRNA 171a sequences to identify SNPs. The 171a mature miRNA and pre-miRNA are indicated by the identifiers mature_miR171a (SEQ ID NO:67) and zma-MIR171a (SEQ ID NO:68), respectively. The wild type B73 sequence is indicated by the identifier, chr4_240118217...240118861 (SEQ ID NO:69). The other corn lines aligned are: IJ6208./1643 (SEQ ID NO.70); A0100871626 (SEQ ID NO:71); BB3004./1644 (SEQ ID NO:72); CE8415./1573 (SEQ ID NO:73); DC4015./1587 (SEQ ID NO:74); FF609672619 (SEQ ID NO:75); PJ7065./1595 (SEQ ID NO:76); WR0588./1570 (SEQ ID NO:77); XF71 1071464 (SEQ ID NO:78); XO574471604 (SEQ ID NO:79); XPCC00371613 (SEQ ID NO:80); and XPFF003./1622 (SEQ ID NO:81).
Figures 3A-3N. Alignment of miRNA 393a sequences to identify SNPs. The mature miRNA and pre-miRNA are indicated by the identifiers mature_miRNA./123 (SEQ ID NO:82) and pre_miRNA./1127 (SEQ ID NO:83), respectively. The wild type
B73 sequence is indicated by the identifier, chr2_736214...736992 (SEQ ID NO:84). The other corn lines aligned are: AO1008./1792 (SEQ ID NO:85); XF7110./1766 (SEQ ID NO:86); FF6096./1757 (SEQ ID NO:87); X05744./1755 (SEQ ID NO:88); ID5829./1612 (SEQ ID NO:89); FSNU505./1739 (SEQ ID NO:90); HT7049HL./1566 (SEQ ID NO:91); AX5707./1763 (SEQ ID NO:92); CC7752./1698 (SEQ ID NO:93); AF4031./1757 (SEQ ID NO:94); PJ7065./1782 (SEQ ID NO:95); HH5982./1566 (SEQ ID NO:96); CE8415./1733 (SEQ ID NO:97); IQ1332./1762 (SEQ ID NO:98); ID261871625 (SEQ ID NO:99); XPFF003./1746 (SEQ ID NO:100); AA3941./1745 (SEQ ID NO:101); WR0588./1758 (SEQ ID NO:102); IJ6208./1765 (SEQ ID NO:103); ID700271758 (SEQ ID NO:104); XPCC003./1670 (SEQ ID NO:105); CC8032./1708 (SEQ ID NO:106); DC4015./1698 (SEQ ID NO:107); and BB3004./1415 (SEQ ID NO:108).
Figure 4. Procedure for phenotypic data analysis for the hybrid panel. There were two purposes for phenotypic data analysis: data quality control and phenotypic adjustment for fitting association statistical models. Note that prior to phenotypic adjustment, there was also a data splitting process to subset the data according to various experimental conditions (e.g. locations, LD panels, and water treatments). The analysis for the inbred panel was similar but much simpler, because there were fewer data splits.
Figure 5 shows the 169g amplicon (SEQ ID NO: 109). The SNPs are denoted with boxes. The pre-miRNA sequence is underlined, and the mature miRNA sequence is underlined and shaded.
Figure 6 shows the 171 amplicon (SEQ ID NO:110). The SNPs are denoted with boxes. The pre-miRNA sequence is underlined, and the mature miRNA sequence is underlined and shaded.
Figure 7 shows the 373 amplicon (SEQ ID ΝΟ. 11). The SNPs are denoted with boxes. The pre-miRNA sequence is underlined, and the mature miRNA sequence is underlined and shaded.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of this specification, unless otherwise indicated, all numbers expressing quantities, conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification are approximations that can vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between, and inclusive of, the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g. 1 to 6.1 , and ending with a maximum value of 10 or less, e.g., 5.5 to 10.
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms described below are more fully explained by reference to the specification as a whole.
It is further noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent.
"Plant" includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
As used herein, the term plant is also used in its broadest sense, including, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonas reinhardtii). Non-limiting examples of plants include plants from the genus Arabidopsis or the genus Oryza. Other examples include plants from the genuses Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia. Still other examples of plants include, but are not limited to, wheat, cauliflower, tomato, tobacco, corn, petunia, trees, etc. As used herein, the term "cereal crop" is used in its broadest sense. The term includes, but is not limited to, any species of grass, or grain plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum, triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes or soybeans, etc.). As used herein, the term "crop" or "crop plant" is used in its broadest sense. The term includes, but is not limited to, any species of plant or algae edible by humans or used as a feed for animals or used, or consumed by humans, or any plant or algae used in industry or commerce.
The term "plant part" includes differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue
or cell culture. The aforementioned term also includes plant products, such as grain, fruits, and nuts.
The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.
The term "genome" refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) present in each cell of an organism, or virus or organelle; (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.
"Progeny" comprises any subsequent generation of a plant. Progeny will inherit, and stably segregate, genes and transgenes from its parent plant(s).
The terms "recombinant construct", "expression construct", "chimeric construct", "construct", and "recombinant DNA construct" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric 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 found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78- 86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.
This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. The construct may be transcribed to form an RNA, wherein the RNA may be capable of forming a double stranded RNA and/or hairpin structure. This construct may be expressed in the cell, isolated, or synthetically produced. The construct may further comprise a promoter, or other sequences that facilitate manipulation or expression of the construct.
As used herein, the terms "suppression", "silencing" or "inhibition" are used interchangeably to denote the down-regulation of the expression of a product of a target sequence relative to its normal expression level in a wild type organism. Suppression includes expression that is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the wild type expression level.
As used herein, "encodes" or "encoding" refers to a DNA sequence that can be processed to generate an RNA and/or polypeptide.
As used herein, "expression" or "expressing" refers to production of a functional product, such as, the generation of an RNA transcript from an introduced construct, an endogenous DNA sequence, or a stably incorporated heterologous DNA sequence. The term may also refer to a polypeptide produced from an mRNA generated from any of the above DNA precursors. Thus, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide).
As used herein, "heterologous" with respect to a 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. For example, with respect to a nucleic acid, it can be a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is modified from its native form in composition and/or genomic locus by deliberate human intervention. In particular, the term heterologous, as used herein, includes single nucleotide polymorphisms that may be introduced into a host organism.
The term "host cell" refers to a cell that contains or into which is introduced a nucleic acid construct and supports the replication and/or expression of the construct. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as fungi, yeast, insect, amphibian, nematode, or mammalian cells. Alternatively, the host cells are monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell.
The term "introduced" means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/ expression 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).
The term "genome" as it applies to a 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.
The term "isolated" refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
As used herein, "microRNA" or "miRNA" refers to an oligoribonucleic acid, which base pairs to a polynucleotide comprising the target sequence causing post- transcriptional regulation by transcript degredation or translational suppression. A "mature miRNA" refers to the miRNA generated from the processing of a "precursor
miRNA" or "pre-miRNA", which is the transcription product from a miRNA template. A "miRNA template" is an oligonucleotide region, or regions, in a nucleic acid construct that encodes the miRNA. The miRNA template may form a double-stranded polynucleotide, including a hairpin structure.
As used herein, "domain" or "functional domain" refers to nucleic acid sequence(s) that are capable of eliciting a biological response in plants. The present invention concerns miRNAs comprised of at least 21 nucleotide sequences acting individually or in concert with other miRNA sequences; therefore a domain could refer to either individual miRNAs or groups of miRNAs. miRNA sequences associated with their backbone sequences could be considered domains useful for processing the miRNA into its active form. As used herein, "subdomains" or "functional subdomains" refer to subsequences of domains that are capable of eliciting a biological response in plants. A miRNA could be considered a subdomain of a backbone sequence. "Contiguous" sequences or domains refer to sequences that are sequentially linked without added nucleotides intervening between the domains.
As used herein, the phrases "target sequence" and "sequence of interest" are used interchangeably. Target sequence is used to mean the nucleic acid sequence that is selected for alteration (e.g., suppression) of expression, and is not limited to polynucleotides encoding polypeptides. The target sequence comprises a sequence that is substantially or fully complementary to the miRNA. The target sequence includes, but is not limited to, RNA, DNA, or a polynucleotide comprising the target sequence. As discussed in Bartel and Bartel ((2003) Plant Phys. 132:709-719), most microRNA sequences are 20 to 22 nucleotides with anywhere from 0 to 3 mismatches when compared to their target sequences.
It is understood that microRNA sequences, such as the 21 nucleotide sequences of the present invention, may still be functional as shorter (20 nucleotide) or longer (22 nucleotide) sequences. In addition, some nucleotide substitutions, particularly at the last two nucleotides of the 3' end of the microRNA sequence, may be useful in retaining at least some microRNA function.
The terms "miRNA 169g," "miRNA 171a," and "miRNA 393" (or "miR169g,"
"miR171a," and "miR393") refer to the respective microRNAs from Zea mays and also
encompass homologous and orthologous microRNAs in other plants. Homologous microRNAs include those with 70% or greater sequence homology to the above-noted miRNAs in Zea mays, for example, at least about 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%. Homologous and orthologous microRNAs will also share a similar chromosomal location.
As understood in the art, the phrase "single nucleotide polymorphism" or "SNP," refers to a single nucleotide variation in a gene or contiguous region upstream or downstream from a gene that differs from the typical genomic sequence for that organism.
A "miRNA region" refers to sequences upstream, downstream, or within a miRNA template that contribute to folding or processing of the miRNA transcript or regulating transcription of the miRNA, i.e., features of the levels, spatial distribution, and/or temporal profile of the miRNA expression. Such miRNA regions can be identified, for example, based upon the presence of at least one single nucleotide polymorphism (SNP) or mutation that enhances or decreases transcript level of a mature miRNA.
As used herein, "nucleic acid" means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms "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 (Cor T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
The term "nucleic acid library" is used to refer to a collection of isolated DNA or RNA molecules that comprise and substantially represent the entire transcribed fraction of a genome of a specified organism or of a tissue from that organism. Construction of
exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references (see, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning— A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
As used herein "operably linked" includes reference to a functional linkage of at least two sequences. Operably linked includes linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
As used herein, "polypeptide" means proteins, protein fragments, modified proteins, amino acid sequences and synthetic amino acid sequences. The polypeptide can be glycosylated or not.
As used herein, "promoter" refers to a nucleic acid fragment, e.g., a region of
DNA, that is involved in recognition and binding of an RNA polymerase and other proteins to initiate transcription. In other words, this nucleic acid fragment is capable of controlling transcription of another nucleic acid fragment.
The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted
to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1 % SDS (sodium dodecyl sulphate) at 37 °C, and a wash in 1x to 2x SSC (20x SSC = 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55 °C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1 % SDS at 37 °C, and a wash in 0.5x to 1x SSC at 55 to 60 °C Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at 37 °C, and a wash in 0.1 x SSC at 60 to 65 °C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA- DNA hybrids, the 7" m can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): 7m = 81.5 °C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500/ L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. 7" m is reduced by about 1 °C for each 1 % of mismatching; thus, 7" m hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 °C Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (7m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1 , 2, 3, or 4 °C lower than the thermal melting point (7m); moderately
stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 °C lower than the thermal melting point (7" m); low stringency conditions can utilize a hybridization and/or wash at 11 , 12, 13, 14, 15, or 20 °C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired 7m those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32 °C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley- Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120, or 240 minutes.
The terms "reliable detection" and "reliably detected" are defined herein to mean the reproducible detection of measurable, sequence-specific signal intensity above background noise.
As used herein, "transgenic" refers to a plant or a cell 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, or heritable, to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression construct. Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extrachromosomal) 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.
As used herein, "vector" refers to a small nucleic acid molecule (plasmid, virus, bacteriophage, artificial or cut DNA molecule) that can be used to deliver a polynucleotide of the invention into a host cell. Vectors are capable of being replicated and contain cloning sites for introduction of a foreign polynucleotide. Thus, expression vectors permit transcription of a nucleic acid inserted therein.
Polynucleotide sequences may have substantial identity, substantial homology, or substantial complementarity to the selected region of the target gene. As used herein "substantial identity" and "substantial homology" indicate sequences that have sequence identity or homology to each other. Generally, sequences that are substantially identical or substantially homologous will have about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity wherein the percent sequence identity is based on the entire sequence and is determined by GAP alignment using default parameters (GCG, GAP version 10, Accelrys, San Diego, Calif.). GAP uses the algorithm of Needleman and Wunsch (Mol. Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of sequence gaps. Sequences which have 100% identity are identical. "Substantial complementarity" refers to sequences that are complementary to each other, and are able to base pair with each other. In describing complementary sequences, if all the nucleotides in the first sequence will base pair to the second sequence, these sequences are fully or completely complementary.
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). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as "dicer." Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., Nature 409:363 2001) and/or pre miRNAs into miRNAs. Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al., Genes Dev. 15:188 2001). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001 , Science 293:834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., Genes Dev. 15:188 2001). In addition, RNA interference can also involve small RNA (e.g., microRNA, or miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences {see, e.g., Allshire, Science 297:1818-1819 2002; Volpe et al., Science 297:1833-1837 2002; Jenuwein, Science 297:2215- 2218 2002; and Hall et al., Science 297:2232-2237 2002). As such, miRNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post- transcriptional level.
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 function, at least in part, 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 (2002) Curr. Biol. 12:735-739; Lau et al., (2001) Science 294:858-862; Lee and Ambros (2001) Science 294:862-864; Llave et al., Plant Cell 14:1605-1619 2002; Mourelatos et al., Genes. Dev. 16:720-728 2002; Park et al., (2002) Curr. Biol. 12:1484-1495; Reinhart et al (2002) Genes. Dev. 16:1616-1626). They are processed from longer precursor transcripts that range in size from approximately 70 to 200 nucleotides, and these precursor transcripts have the ability to form stable hairpin structures. Plants have an enzyme, DCL1 , and evidence indicates that itis involved in processing the hairpin precursors to generate mature miRNAs (Park et al (2002) Curr. Biol. 12:1484-1495; Reinhart et al (2002) Genes. Dev. 16:1616-1626). Furthermore, at least some miRNA hairpin precursors originate as longer polyadenylated transcripts, and several different miRNAs and associated hairpins can be present in a single transcript (Lagos-Quintana et al (2001) Science 294:853-858; Lee et al., (2002) EMBO J. 21 :4663-4670).
MicroRNAs regulate target genes, at least in part, by binding to complementary sequences located in the transcripts produced by these genes. In the case of lin-4 and let-7, the target sites are located in the 3' UTRs of the target mRNAs (Lee et al (1993) Cell 75:843-854; Wightman et al (1993) Cell 75:855-862; Reinhart et al (2000) Nature 403:901-906; Slack et al., Mol. Cell. 5:659-669 2000), and there are several mismatches between the lin-4 and let-7 miRNAs and their target sites. Some studies indicate that binding of the lin-4 or let-7 miRNA may downregulate steady-state levels of the protein encoded by the target mRNA without affecting the transcript itself (Olsen and Ambros, Dev. Biol. 216:671-680 1999). However, in some studies, miRNAs appear to cause specific RNA cleavage of the target transcript within the target site, and that this cleavage step requires 100% complementarity between the miRNA and the target transcript (Hutvagner and Zamore, (2002) Science 297:2056-2060; Llave et al., Plant
Cell 14:1605-1619 2002). miRNAs may contribute to at least two pathways of target gene regulation: Protein downregulation when target complementarity is <100%, and RNA cleavage when target complementarity is 100%. MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nucleotide short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants (Hamilton and Baulcombe 1999; Hammond et al., 2000; Zamore et al., 2000; Elbashir et al., 2001 ), and are incorporated into an RNA- induced silencing complex (RISC) that is similar or identical to that seen for RNAi.
An aspect of the present invention is a method for identifying single nucleotide polymorphisms in miRNA regions using association mapping. Association mapping, including genome-wide association mapping and candidate-gene association mapping, has emerged as a tool to resolve complex trait variation down to the sequence level. Genome-wide association mapping is conducted to find signals of association for various complex traits by surveying genetic variation in the whole genome. Candidate- gene association mapping relates polymorphisms in selected candidate genes that could control phenotypic variation for specific traits. Association mapping relies on chromosomal recombination opportunities over a large number of generations, in the history of a species, which allows the removal of association between a QTL and any marker not tightly linked to it, thus improving the rate of discovery of true association (Jannink and Walsh, Quantitative Genetics, Genomics and Plant Breeding, Kang, Ed. CAB International, (2002) pp. 59-68).
An approach used to link phenotypic variation with genetic loci is marker-trait association (MTA) mapping, also known as linkage disequilibrium (LD) mapping. LD mapping emerged as an important gene mapping tool in early 1990's with the advent of high-throughput genotyping technology, and has been widely used in human genetics to identify genes affecting human diseases. This approach was introduced and began to be adopted in plant gene mapping studies in early 2000's (Flint-Garcia et al. (2003) Annu Rev Plant Biol 54: 357-374). In recent years, success in applying LD mapping has been seen in maize and other crops (Thornsberry et al. (2001) Nat Genet 28: 286- 289).
LD mapping relies on linkage disequilibrium, which is defined as the non-random association of alleles from two different loci (genes or markers) in a natural population. LD mapping assumes that the main cause for LD is linkage that binds loci on the same chromosome together in transmission to next generation. However, due to recombination events accumulated over many generations in a natural population, each chromosome has been shuffled deeply, so that the chromosome has been broken into many tiny regions where loci remain transmitted together, but loci from different regions tend to transmit independently as if they were from different chromosomes. Chromosomal regions where loci are bound together in transmission are commonly known as LD blocks (Reich et al. (2001) Nature 41 1 :199-204). LD mapping identifies genes of interest through genetic markers on the LD blocks where the genes are located. This is done by detecting significant associations between the markers and the traits that the genes affect with a sample of unrelated individuals or a sample of unrelated pedigrees that are genotyped on a selected set of markers covering candidate gene regions or the whole genome, and phenotyped on a set of traits of interest.
Compared with traditional linkage mapping methods that are typically based on artificial biparental segregating populations (e.g., F2, BC, DH, RIL, etc.), LD mapping generally produces better mapping resolution, because of the smaller sizes of LD blocks. In addition, LD mapping is useful in identifying more than two functional alleles at associated markers in a germplasm. Further, LD mapping is efficient for evaluating natural populations.
Linkage disequilibrium may be caused by factors other than linkage, such as mutation, migration, inbreeding, and genetic drift, inter alia. Consequently, LD mapping can be prone to false positives or spurious MTAs. Spurious MTAs are marker-trait associations between unlinked or distantly linked loci. Another consideration is the sample population structure. Population structure has been has been studied extensively, and effective statistical approaches have been developed to significantly reduce false positives in human genetics and in plants as well (Yu et al. (2006) Nat. Genet. 38:203-208). In addition, LD mapping requires high-density marker coverage on the genome in order to capture as many tiny LD blocks as possible. This issue has been largely overcome by high-throughput genotyping technology. However, other
considerations in experimental design include precision and accuracy of phenotype acquisition in addition to throughput (Myles et al. (2009) Plant Cell 21 :2194-2202).
Markers selected for association mapping are often chosen randomly with the goal of having the greatest number of markers spaced evenly across the genome. Another strategy, known as candidate gene strategy, is to make markers to score the alleles of genes that are suspected to influence the phenotype that one will evaluate. The present application discloses a third strategy (i.e., using markers to distinguish alleles of miRNAs that are associated with trait of interest). This third strategy has the advantage that miRNAs regulate many genes, and the genes they regulate often regulate many other genes. The advantages of this strategy are evident based on the findings provided herein: In an association study of 3072 random loci, 101 candidate gene loci and 3 microRNA loci, random loci showed 260 associations (8%), the candidate gene loci showed 41 associations (41 %) and the miRNA loci had 3 associations (100%).
Another aspect of the invention is methods for suppressing a target sequence.
The methods employ any constructs in which a miRNA is designed to identify a region of the target sequence, and inserted into the construct. One can selectively regulate the target sequence by encoding a miRNA having substantial complementarity to a region of the target sequence. The miRNA is provided in a nucleic acid construct which, when transcribed into RNA, is predicted to form a hairpin structure which is processed by the cell to generate the miRNA, which then suppresses expression of the target sequence. Upon introduction into a cell, the miRNA produced suppresses expression of the targeted sequence. The target sequence can be an endogenous plant sequence, or a heterologous transgene in the plant. In particular, the invention includes constructs comprising one or more of SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
The methods provided can be practiced in any organism in which a method of transformation is available, and for which there is at least some sequence information for the target sequence, or for a region flanking the target sequence of interest. It is also understood that two or more sequences could be targeted by sequential transformation, co-transformation with more than one targeting vector, or the construction of a DNA construct comprising more than one miRNA sequence. The
methods of the invention may also be implemented by a combinatorial nucleic acid library construction in order to generate a library of miRNAs directed to random target sequences. The library of miRNAs could be used for high-throughput screening for gene function validation.
General categories of sequences of interest include, for example, those genes involved in regulation or information, such as zinc fingers, transcription factors, homeotic genes, or cell cycle and cell death modulators, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. Other categories of target sequences include genes affecting agronomic traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest also included those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting, for example, kernel size, sucrose loading, and the like. The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality, and quantity of essential amino acids, and levels of cellulose.
For example, genes of the phytic acid biosynthetic pathway could be suppressed to generate a high available phosphorous phenotype. See, for example, phytic acid biosynthetic enzymes including inositol polyphosphate kinase-2 polynucleotides, disclosed in PCT International Publication No. WO 02/059324, inositol 1 ,3,4- trisphosphate 5/6-kinase polynucleotides, disclosed in PCT International Publication No. WO 03/027243, and myo-inositol 1 -phosphate synthase and other phytate biosynthetic polynucleotides, disclosed in PCT International Publication No. WO 99/05298. Genes in the lignification pathway could be suppressed to enhance digestibility or energy availability. Genes affecting cell cycle or cell death could be suppressed to affect growth or stress response. Genes affecting DNA repair and/or recombination could be suppressed to increase genetic variability. Genes affecting flowering time could be suppressed, as well as genes affecting fertility. Any target sequence could be suppressed in order to evaluate or confirm its role in a particular trait or phenotype, or to dissect a molecular, regulatory, biochemical, or proteomic pathway or network.
Target sequences further include coding regions and non-coding regions such as promoters, enhancers, terminators, introns and the like, which may be modified in order
to alter the expression of a gene of interest. For example, an intron sequence can be added to the 5' region to increase the amount of mature message that accumulates (see, e.g., Buchman and Berg, (1988) Mol. Cell. Biol. 8:4395-4405; and Callis et al (1987) Genes Dev. 1 :1 83-1200).
The target sequence may be an endogenous sequence, or may be an introduced heterologous sequence, or transgene. For example, the methods may be used to alter the regulation or expression of a transgene, or to remove a transgene or other introduced sequence such as an introduced site-specific recombination site. The target sequence may also be a sequence from a pathogen, for example, the target sequence may be from a plant pathogen such as a virus, a mold or fungus, an insect, or a nematode. A miRNA could be expressed in a plant that, upon infection or infestation, would target the pathogen and confer some degree of resistance to the plant.
A number of promoters can be used, these promoters can be selected based on the desired outcome. It is recognized that different applications will be enhanced by the use of different promoters in plant expression cassettes to modulate the timing, location and/or level of expression of the miRNA. Such plant expression cassettes may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Constitutive, tissue-preferred or inducible promoters can be employed. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1 '- or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenate promoter (U.S. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter and other transcription initiation regions from various plant genes known to those of skill. If low level expression is desired, weak promoter(s) may be used. Weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (PCT International Publication No. WO 99/43838 and U.S. Patent No. 6,072,050), the core 35S CaMV
promoter, and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Patent No. 6,177,611.
Examples of inducible promoters are the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the ln2-2 promoter which is safener induced (U.S. Patent No. 5,364,780), the ERE promoter which is estrogen induced, and the Axigl promoter which is auxin induced and tapetum specific but also active in callus (PCT US01/22169).
Examples of promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. An exemplary promoter is the anther specific promoter 5126 (U.S. Patent Nos. 5,689,049 and 5,689,051). Examples of seed preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A. et al. (1986) Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21 ):6426; and Kloesgen, R. B. et al. (1986) Mol. Gen. Genet. 203:237-244. Promoters that express in the embryo, pericarp, and endosperm are disclosed in U.S. Patent No. 6,225,529 and PCT International Publication No. WO 00/12733.
In some aspects it will be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1 ,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:1 11-116. See also PCT International Publication No. WO 99/43819.
Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331 ; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427- 2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See
also, Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91 :2507-251 1 ; Warner et al. (1993) Plant J. 3:191-201 ; Siebertz et al. (1989) Plant Cell 1 :961-968; U.S. Patent No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant. Path. 41 :189-200).
Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the constructions of the polynucleotides. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotech. 14:494-498); wunl and wun2, U.S. Patent No. 5,428,148; winl and wing (Stanford et al. (1989) Mol. Gen. Genet. 215:200- 208); systemin (McGurl et al. (1992) Science 225:1570- 1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783- 792; Eckelkamp et al. (1993) FEBS Lett. 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid steroid-responsive promoters (see, for example, the glucocorticoid- inducible promoter in Schena et al. (1991) Proc. ■ Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline- repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156.
Tissue-preferred promoters can be utilized to target enhanced expression of a sequence of interest within a particular plant tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7)792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 1 12(3):1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1 129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, e.g., Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. In addition, the promoters of cab and rubisco can also be used. See, e.g., Simpson et al. (1958) EMBO J. 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051 -1061 (root specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):1 1-22 (full- length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633- 641 , where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing non legume Parasponia andersonii and the related non-nitrogen fixing non legume Trema tomentosa are described. The promoters of these genes were linked to a 13-glucuronidase reporter gene and introduced into both the non legume
Nicotians tabacum and the legume Lotus corniculatus, and in both instances root- specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue- preferred DNA determinants are dissociated in those promoters. Teen et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TRV gene, fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root- preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4) :681-691. See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459, 252; 5,401 ,836; 5,110,732; and 5,023,179. The phaseolin gene (Murai et al. (1983) Science 23:476-482 and Sengopta-Gopalen et al. (1988) PNAS 82:3320-3324.
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing the DNA construct include microinjection (Crossway et al. (1986) Biotechniques 4:320-334; and U.S. Patent No. 6,300,543), sexual crossing, electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-med ated transformation (Townsend et al., U.S. Patent No. 5,563,055; and U.S. Patent No. 5,981 ,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Patent No. 4,945,050; Tomes et al., U.S. Patent No. 5,879,918; Tomes et al., U.S. Patent No. 5,886,244; Bidney et al., U.S. Patent No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). See also Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology
5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et al., U.S. Patent Nos. 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440- 444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311 :763-764; Bowen et al., U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560- 566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); and U.S. Patent No. 5,736,369 (meristem transformation).
The nucleotide constructs may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that useful promoters encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, e.g., U.S. Patent Nos. 5,889,191 , 5,889,190, 5,866,785, 5,589,367 and 5,316,931.
DNA constructs containing miRNA genes and their corresponding upstream and downstream regulatory regions may be integrated of the into the host cell chromosome according to conventional methods, e.g., by homologous recombination or other methods of integration, including targeted integration at a particular host chromosomal site.
In some aspects, transient expression may be desired. In those cases, standard transient transformation techniques may be used. Such methods include, but are not limited to viral transformation methods, and microinjection of DNA or RNA, as well other methods well known in the art.
The cells from the plants that have stably incorporated the nucleotide sequence may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic imparted by the nucleotide sequence of interest and/or the genetic markers contained within the target site or transfer cassette. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
Initial identification and selection of cells and/or plants comprising the DNA constructs may be facilitated by the use of marker genes. Gene targeting can be performed without selection if there is a sensitive method for identifying recombinants, for example if the targeted gene modification can be easily detected by PCR analysis, or if it results in a certain phenotype. However, in most cases, identification of gene targeting events will be facilitated by the use of markers. Useful markers include positive and negative selectable markers as well as markers that facilitate screening, such as visual markers. Selectable markers include genes carrying resistance to an antibiotic such as spectinomycin (e.g. the aada gene, Svab et al. 1990 Plant Mol. Biol. 14:197), streptomycin (e.g., aada, or SPT, Svab et al. 1990 Plant Mol. Biol. 14:197; Jones et al. 1987 Mol. Gen. Genet. 210:86), kanamycin (e.g., nptll, Fraley et al. 1983 PNAS 80:4803), hygromycin (e.g., HPT, Vanden Elzen et al. 1985 Plant Mol. Biol. 5:299), gentamycin (Hayford et al. 1988 Plant Physiol. 86:1216), phleomycin, zeocin, or bleomycin (Hille et al. (1986) Plant Mol. Biol. 7:171), or resistance to a herbicide such as phosphinothricin (bar gene), or sulfonylurea (acetolactate synthase (ALS)) (Charest et al. (1990) Plant Cell Rep. 8:643), genes that fulfill a growth requirement on an incomplete media such as HIS3, LEU2, URA3, LYS2, and TRP1 genes in yeast, and
other such genes known in the art. Negative selectable markers include cytosine deaminase (codA) (Stougaard (1993) Plant J. 3:755-761 ), tms2 (DePicker et al. (1988) Plant Cell Rep. 7:63-66), nitrate reductase (Nussame et al. (1991) Plant J. 1 :267-274), SU1 (O'Keefe et al. (1994) Plant Physiol. 105:473-482), aux-2 from the Ti plasmid of Agrobacterium, and thymidine kinase. Screenable markers include fluorescent proteins such as green fluorescent protein (GFP) (Chalfie et al. (1994) Science 263:802; U.S. Patent No. 6,146,826; U.S. Patent No. 5,491 ,084; and PCT International Publication No. WO 97/41228), reporter enzymes such as 13-glucuronidase (GUS) (Jefferson R. A. (1987) Plant Mol. Biol. Rep. 5:387; U.S. Patent No. 5,599,670; and U.S. Patent No. 5,432,081), 13-galactosidase (lacZ), alkaline phosphatase (AP), glutathione S- transferase (GST) and luciferase (U.S. Patent No. 5,674,713; and Ow et al. (1986) Science 234(4778):856-859), visual markers like anthocyanins such as CRC (Ludwig et al. (1990) Science 247(4841 ):449-450) R gene family (e.g., Lc, P, S), A, C, R-nj, body and/or eye color genes in Drosophila, coat color genes in mammalian systems, and others known in the art.
One or more markers may be used in order to select and screen for gene targeting events. One common strategy for gene disruption involves using a target modifying polynucleotide in which the target is disrupted by a promoterless selectable marker. Since the selectable marker lacks a promoter, random integration events generally do not lead to transcription of the gene. Gene targeting events will put the selectable marker under control of the promoter for the target gene. Gene targeting events are identified by selection for expression of the selectable marker. Another common strategy utilizes a positive-negative selection scheme. This scheme utilizes two selectable markers, one that confers resistance (R+) coupled with one that confers sensitivity (S+), each with a promoter. When this polynucleotide is randomly inserted, the resulting phenotype is R+/S+. When a gene targeting event is generated, the two markers are uncoupled and the resulting phenotype is R+/S-. Examples of using positive-negative selection are found in Thykjer et al. (1997) Plant Mol. Biol. 35:523- 530; and PCT International Publication No. WO 01/66717.
Another aspect of the invention concerns a plant, cell, and seed comprising the construct and/or the miRNA. Typically, the cell will be a cell from a plant, but other
prokaryotic or eukaryotic cells are also contemplated, including but not limited to viral, bacterial, yeast, insect, nematode, or animal cells. Plant cells include cells from monocots and dicots. The invention also provides plants and seeds comprising the construct and/or the miRNA.
EXAMPLES
The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.
Example 1
Identification and Analysis of SNP Diversity in miRNA Regulatory Regions of Three miRNAs from Inbred Maize Lines
Genomic DNA amplicons containing the miR169g, miR171a, and miR393 regions and the upstream and downstream flanking sequences were amplified using the primers shown in Table 1 from a Maize genomic DNA library derived from a diverse panel of inbred lines. SNPs were identified by aligning the sequences from the Maize lines using SeqScape Software Version 2.5 from Applied Biosystems (Figures 1A-1 P, 2A-2L, and 3A-3N).
Genotyping LD Mapping Panels
Using the putative SNPs identified in Example 1 as a guide, a TAQMAN® genotyping assay (Applied Biosystems) was developed to evaluate the prevalence of SNPs in the three miRNAs, miR171 and miR393 regions on approximately 700 base pair amplicons (Livak et al. (1995) Nat. Genetics 9:341-342). In allelic discrimination assays, a PCR assay includes a forward and reverse primer and a specific, fluorescent, dye-labeled probe for each of two alleles. The probes contain different fluorescent reporter dyes (VIC® and FAM, or TET and FAM) to differentiate the amplification of each allele. FAM is 6-carboxyfluoroscein, TET is 6-carboxy-4,7,2',7'- tetrachlorofluorescein, and VIC® is a proprietary dye (Applied Biosystems). A non- fluorescent quencher on each probe suppresses the fluorescence until amplification by PCR. During PCR, each probe anneals specifically to complementary sequences between the forward and reverse primer sites. Taq DNA polymerase then cleaves the probes that are hybridized to each allele. Cleavage separates the reporter dye from the quencher, which results in increased fluorescence by the reporter dye. Thus, the fluorescent signals generated by PCR amplification indicate that one or both alleles are present in the sample. In addition to the nonfluorescent quencher, the probe also contains a minor groove binder at the 3' end, which results in an increased melting temperature (7" m), thereby allowing high specificity with the use of shorter oligos. These probes therefore exhibit greater 7m differences when hybridized to matched and mismatched templates, which provides more accurate allelic discrimination. Probes of this type can be manufactured at either ABI (MGB™ quencher) or Biosearch Technologies (BHQPLUS™ quencher). At the end of PCR thermal cycling, fluorescence of the two reporter dyes is measured on an ABI 7900 Sequence Detection System. An increase in fluorescence for one dye indicates homozygosity for the corresponding allele. Increase in both fluorescent signals indicates heterozygosity.
Table 2: TAQMAN® Primers and Probes
Probe
Star Fluorophore
F/ Primer or Prob Sequence
Primer Name t SEQ ID , Quencher,
R (all are 5'→3')
Pos. Groove
Binder*
169F2_169gR2-mi RNA169g_127(1)
SEQ ID
SM1480DQF1 F 83 G AG ATTG CG CGAATCAGTC A —
NO:1 1
SEQ ID C I GC I GCA I I I GCCG I I I A I G
SM1480DQR1 R 160 —
NO:12 AG
SEQ ID FAM, BHQ,
SM1480DQA1 FM F 1 16 AC GTGTG GAG C CTTT
NO:13 BGB
SEQ ID TET, BHQ,
SM1480DQA2TT F 1 16 ACGTGTGGAGCTTTTC
NO:14 BGB
169F2_169gR2-mi RNA169g_213(1)
SEQ ID CTCATAAACGGCAAATGCAG
SM1480BQF1 F 138 —
NO:15 CAG
SEQ ID ACGCACGTCGGTCTACCACA
SM1480BQR1 R 247 —
NO:16 T
SEQ ID TET, BHQ,
SM1480BQA2TT F 198 TTGGTAATCAGTATCTGG
NO:17 BGB
SEQ ID FAM, BHQ,
SM1480BQA1 FM F 202 TAATCAGTATCCGGGAA
NO:18 BGB
169F2_169gR2-mi RNA169g_670(1)
SEQ ID
SM1480AQR1 R 712 ATGAGCCAGCTGATGA —
NO:19
SEQ ID
SM1480AQF1 F 551 G AAG G C CTCTTCTTCTC —
NO:20
SEQ ID FAM, BHQ,
SM1480AQA1 FM R 680 ACAGCCATACATACCT
NO:21 BGB
SEQ ID TET, BHQ,
SM1480AQA2TT R 680 ACAGCCATACTTACCT
NO:22 BGB
171f1_171r1-miRNA171a_44 5(1)
SEQ ID TCCACCATAAGTTTACACACA
SM1479BQF1 F 382 —
NO:23 GAG
SEQ ID GGCACAGAGGGAGTATAATA
SM1479BQR1 R 499 —
NO:24 GACA
SEQ ID FAM, BHQ,
SM1479BQA1 FM F 435 AG GTTAG AC CACTCGTT
NO:25 BGB
SEQ ID TET, BHQ,
SM1479BQA2TT F 434 AAG GTTAG AC C AGTC GTT
NO:26 BGB
393f2_393r2-miRNA39 3 _ 152(1)
SEQ ID GCAACAGCCATCATCGTCATT
SM1481AQF1 F 11 1 —
NO:27 C
SEQ ID
SM1481AQR1 R 256 CAGCTGGGAGGAAGGGAAA —
NO:28
SEQ ID FAM, BHQ,
SM1481AQA1 FM F 144 CCATCATCCTCGTCT
NO:29 BGB
SEQ ID TET, BHQ,
SM1481AQA2TT F 144 CCATCATCGTCGTCT
NO:30 BGB
393f2_393r2-miRNA39 3 _ 213(1)
SEQ ID
SM1481 BQF1 F 0 CTGGGAGGAAGGGAAA —
NO:31
SEQ ID
SM1481 BQR1 R 0 ACAGCCATCATCGTCATTC —
NO:32
SEQ ID TET, BHQ,
SM1481 BQA2TT F 0 CGAGGTCGTAGCCA
NO:33 BGB
SEQ ID FAM, BHQ,
SM1481 BQA1 FM F 0 CGAGGACGTAGCCA
NO:34 BGB
393f2_393r2-miRNA39 3 _ 629(1)
SEQ ID
SM1481 CQF1 F 601 TCGCCTACTTGCTCTC —
NO:35
SEQ ID
SM1481 CQR1 R 724 GCTCCCATGAGCAAATTG —
NO:36
SEQ ID TET, BHQ,
SM1481 CQA2TT F 622 ACGTACTGGCTACATC
NO:37 BGB
SEQ ID FAM, BHQ,
SM1481 CQA1 FM F 617 CACGTACGTACTAGCT
NO:38 BGB
393f2 _393r2-miRNA3 i3_782(1)
SEQ ID
SM1481 DQF1 F 0 GCAGACAAGTACAAACATAG —
NO:39
SEQ ID
SM1481 DQR1 R 0 ACGATGAGCGAAAGGAAA —
NO:40
SEQ ID TET, BHQ,
SM1481 DQA2TT F 0 AAATAGCTGCCGATTCAT
NO:41 BGB
SEQ ID FAM, BHQ,
SM1481 DQA1 FM F 0 TAGCTGCCGATTAATTC
NO:42 BGB
*FAM is 6-carboxyf uoroscein; TET is 6-carboxy-4,7,2',7'-tetrachlorofluorescein; BHQ is Black Hole Plus QU ENCHER®; BGB is BioSource Groove Binder
To validate TAQMAN® allelic discrimination assays for association with drought tolerance, plants were selected based on their known phenotypic status and compared to the genotype at the specific SNP location. DNA was extracted from leaf tissue of seedlings 7-10 days after planting. DNA can be extracted from plant tissue in a variety of ways, including the CTAB method, sodium hydroxide, and the Dellaporta method. DNA is diluted in TE buffer (10 mM Tris HCI, pH 7.5, 1 mM EDTA) and stored at 4°C until used in PCR reactions. PCR reactions were set up in 5 pL final volumes according to Table 3.
PCR plates were placed in ABI 9700 Thermal cyclers and the following thermocycle programs were run.
Table 4: TAQMAN® Thermocycle Programs
Task SNP1
Initial denaturation 50 °C for 2 min.
— 95 °C for 10 min.
Cycles 95 °C for 15 sec.
— 60 °C for 1 min.
Number of cycles 40
Final elongation 72 °C for 5 min.
Hold at 4 °C Indefinite
The ABI 7900 Sequence Detection System, or "TAQMAN®" was used to visualize the results of an allelic discrimination SNP assay. Using the Sequence Detection System (SDS, Applied Biosystems) software, allele calls were determined based on the fluorescence for the two dyes measured in each sample. Table 5 shows the SNP positions and allele types for amplicons 169g, 393, and 171a.
Table 5
Locus
Table 6 is the summary of haplotypes observed in plants and the number of occurrences.
Table 6
SMI 479 1 A T:C SM1479AQ:SM1479BQ 698
SMI 479 2 B T:G SM1479AQ:SM1479BQ 267
SM1479 3 C C.C SM1479AQ:SM1479BQ 51
SM1479 4 D C:G SM1479AQ:SM1479BQ 79
Total 1095 miR A393
SM1481 1 A C:A:A:A SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 189
SM1481 2 B C:A:A:G SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 136
SM1481 3 C C:A:G:G SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 440
SM1481 4 D C:T:A:A SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 12
SM1481 5 E C:T:G:G SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 2
SM1481 6 F G:T:A:A SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 1
SM1481 7 G G:T:A:G SM1481AQ:SM1481BQ:SM1481CQ:SM1481DQ 149
Total 929 miRNA169
SM1480 1 A A:C:C SM1480AQ:SM1480BQ:SM1480DQ Hltapoype 3
SM1480 2 B A:C:T SM1480AQ:SM1480BQ:SM1480DQ 65 F (#)rea4
SM1480 3 C T:C:T SM1480AQ:SM1480BQ:SM1480DQ 79
SM1480 4 D T:T:T SM1480AQ:SM1480BQ:SM1480DQ 328
Total 1064
Example 3
Marker-Trait Association Analysis of miRNAs from Inbred and Hybrid Maize
An association mapping study begins with development of a population sample, continues with genotyping and phenotyping all individuals in the sample, and ends with data analysis and result summary. The population sample is a set of unrelated individuals (with no known pedigree relationships), which is called the linkage disequilibrium (LD) panel, or a set of unrelated pedigrees (Cardon and Bell (2001) Nat. Rev. Genet. 2:91-99). An association study needs to make many strategic decisions
around the population sample, genetic markers, genotyping platform, experimental design (e.g. treatments, locations and repetitions) for phenotyping with field trials, and the choice of appropriate statistical procedure and methods. The reliability and applicability of MTA results from the study depend heavily on the size and composition of the population sample, genomic coverage of genetic markers (candidate-genes based or genome-wide), precision of genotyping and phenotyping, and appropriate use of statistical procedure and methods.
The population samples used in this study were from two commercially establish LD panels of diverse inbred lines, an inbred maize panel and a hybrid maize panel. The hybrid panel further consisted of two subpanels: the non-stiff stalk (NSS) panel and the stiff stalk (SS) panel, while the inbred panel is a mixture of both SS and NSS inbreds. NSS and SS are the two main targeted heterotic groups in maize. The inbred panel and both hybrid subpanels each consisted of approximately 600 inbred lines selected from a platform of 2,075 inbreds that represent the wide genetic diversity and maturity groups (early, intermediate, and late) in the maize germplasm.
The inbred panel was genotyped and phenotyped directly using the inbred panel lines. The hybrid panel was genotyped on the inbred panel as well, and phenotyping was conducted on the hybrids of the inbred panel with a commercially important inbred as the tester. The combination of phenotypic data on both inbreds and hybrids was intended to study the effects of genetic backgrounds (homozygous and heterozygous) on MTAs.
The two LD panels were each phenotyped in one year at multiple locations. Two water treatments were assessed; normal irrigation (WET) and flowering-time drought stress (DRY) were conducted with both panels. These experiments assessed the effects of MTAs on yield and drought tolerance under different irrigation conditions.
After phenotyping, WET and DRY treatments were applied to the inbred maize panel. The first location had 5 repetitions for DRY treatment and 2 repetitions for WET treatment, while the second location had 6 DRY repetitions and 3 WET repetitions. The arrangement of the repetitions in the field was based on maturity groups (early, intermediate, and late) to control for field differences.
After phenotyping, each subpanel of the hybrid maize panel (SS or NSS) was grown at 5 locations with WET treatment, and 3 locations with DRY treatment. Three repetitions were applied for WET treatment, and 6 repetitions for DRY treatment, at all locations where the treatment was applied.
The field trials were specially selected as managed stress environments to permit effective water treatments, in particular the DRY treatment. In these trials, the use of more DRY repetitions reduced the standard errors in phenotypic observations under drought conditions.
A total of ~30 yield and physiological/morphological traits were directly observed and/or calculated for the two LD panels. However, the trait sets used for each panel were very different. The inbred panel was typed using more traits, including yield and its components, several physiological/morphological traits, and drought response traits. By comparision, no yield component traits or drought response traits were typed with hybrid panel. The focus of the hybrid panel was on yield productivity, while the inbred panel was examined to identify novel genes acting on agronomic traits.
There were two purposes for phenotypic data analysis: data quality control (QC) and phenotypic adjustment for fitting association statistical models. The procedure for analyzing the phenotypic data on the hybrid panel is shown in the flowchart in Figure 4. The phenotypic data were split, according to various experimental conditions, in order to detect MTAs that might be caused by various types of gene by environment interactions. 938 lines (434 NSS, 504 SS) were phenotyped for 13 trait in DRY and WET conditions. Data splitting was carried out prior to phenotypic adjustment for model fitting. It was intended to subset the cleaned data according to various experimental conditions including water treatments. Data for each split was then analyzed separately to detect MTAs under particular experimental conditions to capture effects from G*E and GxG interactions.
Six splits were created for the inbred panel data, three for each location, including two splits for DRY and WET and one split combining data from the two treatments. Data splitting for the hybrid panel was much more complicated, which split the data for water treatments, location groups, LD panels, and important combinations between water treatments and panels. In total, there were 83 splits for the hybrid panel.
Note that location groups for the hybrid panel were determined based on similarity among locations in maize growing environments and trait responses using genotype main effect plus genotype by environment interaction (GGE) biplot analysis. In order to fit the statistical models for association analysis, split-specific phenotypic adjustment was done to remove all non-genetic effects (or design-of-experiment (DOE) effects), including effects from locations, repetitions, LD panels, water treatments, etc., depending on the data split in question. At the end of this process, a breeding value or overall genetic effect for each trait was calculated for each inbred in the split. Example 4
Evaluation of Phenotypic Adjustment
Phenotypic data adjustment is a necessary step for fitting the GLM / MLM association models. However, phenotypic adjustment was conducted with MLM, which relies on a few statistical assumptions, including independency between fitted values and random residuals, and normal distribution for random residuals. Violation of these assumptions would affect the reliability and accuracy of the final MTA results (p values, etc.). Therefore, it was important to determine the quality (model fitness) of the adjusted phenotypic data, so that the MTA results from the adjusted data would not be over-interpreted.
After adjusting phenotypic data, two plots were also outputted from phenotypic adjustment for each data split. The first plot fitted values against model residuals, which shows the independency between fitted values and residuals. The second plot was a QQ plot, which indicates normality of the distribution. A 3-level scoring method was used to visually evaluate the quality of the adjusted data. For good-level data, there was a roughly rectangle distribution of data points, suggesting a good independency of residual distribution from fitted values. Furthermore, the data points were mostly on the diagonal line of the QQ plot, which is expected for normal residuals. For bad-level fitness, both plots showed large deviation from the expected values, and third level fitness was in between the good and bad levels.
With this scoring system, all of the eleven main data splits for the hybrid LD panel were assessed. Grain moisture traits (GMSAP and GMSTP) and grain yield traits
(YGSMN, YGSAN, YGSMN/GMSTP, and YGSAN/GMSAP) all had good model fitness in phenotypic adjustment. However, two yield traits unadjusted for standard moisture (YGHMN and YGHAN) did not have very good fitness in phenotypic adjustment. Morphological traits (ERHTN and PLHTN), and flowering time traits (SLK5N, ASIDN, and POL5N) had fair model fitness. In addition, four traits (BRRNN, STD_N, STKLN, and STKLP) had bad fitness in all the relevant data splits. These traits were not analyzed with GLM/MLM for associations. Table 7 shows the effect of a single allele on a particular plant trait for 24 MTAs that passed Bonferroni correction cutoff threshold in hybrid panel.
Looking at the first row of Table 7 and cross-referencing Table 5, one can see that the SNP at position 701 of the 169g amplicon (i.e., marker SM1480AQ) is associated with grain moisture adjusted percentage (GMSAP). Specifically, plants with the "T" allele have 0.44% less moisture at harvest. Plants possessing this allele are therefore more desirable than those with the "A" allele, as grain stores better at lower moisture percentage.
In a similar fashion, looking at the third row from the bottom of Table 7, one also sees that that the "T" allele is also associated with grain yield at harvest moisture percentage. Specifically, plants with the "T" allele yield 0.9 bushels per acre less than those with the "A" allele at harvest moisture percentage. This relationship between grain moisture percentage and grain yield at harvest moisture percentage is typical.
Table 8 is similar to Table 7, but cross-references Table 6 and shows the effect of haplotype on a particular plant trait. For example, looking at row 23, one sees that marker SM1480 is associated with grain moisture adjusted percentage (GMSAP), consistent with the first row of Table 7 discussed above. As shown in Table 7, four combinations of alleles (out of eight possible) in the SM1480 marker are present in the 1064 plants examined. The most frequent haplotype resulting in this favorable phenotype is the "C" haplotype (i.e., a "T" at position 174 of the 169g amplicon, a "C" at position 259 of the 169g amplicon, and a "T" at position 701 of the 169g amplicon), while the most frequent haplotype resulting in an unfavorable phenotype is the "A" haplotype (i.e., a "A" at position 174 of the 169g amplicon, a "C" at position 259 of the 169g amplicon, and a "C" at position 701 of the 169g amplicon). The effect of the "C" haplotype on grain moisture adjusted percentage ranges from 0.84279 to 1.5428 and the mean is 1.18, meaning that plants having these variant alleles have 1.18% less moisture at harvest (which is desirable for the reasons described above).
Table 8
19 169 DSFLR2 SM1480 A D 0.6178 0.62
20 DSFLR3 SM1480 C A 0.5707 0.57
21 EARPN SM1480 D A 0.1712 0.17
22 ERHTN SM1480 D A 2.711- 4.39
6.3184
23 GMSAP SM1480 C A 0.84279- 1.18
1.5428
24 GMSTP SM1480 C A 0.96-2.1841 1.41
25 KRLNN SM1480 D C 0.6891 0.69
26 POL5N SM1480 A D 1.05 1.05
27 SLK5N S 1480 A D 1.42 1.42
28 YGHAN SM1480 B C 5.98 5.98
29 YGhMN SM1480 B C 9.09 9.09
30 YGSAN SM1480 B C 3.07 3.07
31 YGSAN/GMSAP SM1480 C A 0.47-0.79 0.65
32 YGSMN SM1480 B C 5.38-18.74 12.06
33 YGSMN/GMSTP SM1480 C A 0.71-0.86 0.79
34 miRNA ASIDN SM1479 C B 0.097-0.102 0.10
35 171 ERHTN SM1479 B C 1.74-4.27 2.75
36 GMSAP SM1479 C B 0.84-1.26 0.97
37 GMSTP SM1479 C B 0.98-1.26 1.16
38 KRRWN SM1479 B C 0.1608- 0.40
0.6392
39 PLHTN SM1479 D B 2.13-4.27 3.16
40 SLK5N SM1479 C B 0.40-0.51 0.46
41 YGHAN SM1479 B C 3.98-6.40 5.19
42 YGhMN SM1479 A C 4.99-5.68 5.26
43 YGSAN SM1479 A D 2.49-3.92 3.20
44 YGSAN/GMSAP SM1479 C D 0.18-0.30 0.71
45 YGSMN SM1479 A D 3.51-4.72 4.11
46 YGSMN/GMSTP SM1479 C D 0.21-0.32 0.27
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.
Claims
1. A method of identifying a single nucleotide polymorphism associated with a plant trait, the method comprising:
(a) identifying a microRNA region of a plant genome associated with the plant trait;
(b) sequencing the microRNA region in at least two lines of a plant species, wherein at least one of the at least two lines possesses the plant trait and at least one of the at least two lines does not possess the plant trait;
(c) identifying at least one single nucleotide polymorphism in the microRNA region of the at least two lines; and
(d) correlating the at least one single nucleotide polymorphism in the microRNA region with the plant trait.
2. The method of claim 1 , wherein the at least one single nucleotide polymorphism is located in a flanking sequence portion of the microRNA region.
3. The method of claim 3, wherein the flanking sequence portion of the microRNA region flanks a mature miRNA portion of the microRNA region comprising a sequence selected from the group consisting of SEQ ID NOs: 43, 67 and 82.
4. The method of claim 2, wherein the flanking sequence portion of the microRNA region flanks a pre-miRNA portion of the microRNA region comprising a sequence selected from the group consisting of SEQ ID NOs: 44, 68 and 83.
5. The method of claim 4, wherein the flanking sequence portion of the microRNA region is selected from the group consisting of
(a) nucleotides 1 - 490 of SEQ ID NO: 45;
(b) nucleotides 630 - 815 of SEQ ID NO: 45;
(c) nucleotides 1 - 273 of SEQ ID NO: 69; (d) nucleotides 373 - 645 of SEQ ID NO: 69;
(e) nucleotides 1 - 303 of SEQ ID NO: 84; and
(f) nucleotides 429 - 779 of SEQ ID NO: 84.
6. The method of claim 5, wherein the at least one single nucleotide polymorphism is located at a nucleotide corresponding to a position selected from the group consisting of:
(a) position 216 of SEQ ID NO: 45, wherein the nucleotide is a C;
(b) position 301 of SEQ ID NO: 45, wherein the nucleotide is a C;
(c) position 743 of SEQ ID NO: 45, wherein the nucleotide is a T;
(d) position 444 of SEQ ID NO: 69, wherein the nucleotide is a C;
(e) position 500 of SEQ ID NO: 69, wherein the nucleotide is a C;
(f) position 120 of SEQ ID NO: 84, wherein the nucleotide is a C;
(g) position 218 of SEQ ID NO: 84, wherein the nucleotide is an A;
(h) position 575 of SEQ ID NO: 84, wherein the nucleotide is an A; and
(i) position 693 of SEQ ID NO: 84, wherein the nucleotide is an A.
7. The method of claim 1 , wherein the at least one single nucleotide polymorphism is located in a pre-miRNA portion of the microRNA region.
8. The method of claim 7, wherein pre-miRNA portion of the microRNA region comprises a sequence selected from the group consisting of SEQ ID NOs: 43, 67 and 82.
9. The method of claim 7, wherein pre-miRNA portion of the microRNA region comprises a sequence selected from the group consisting of SEQ ID NOs: 44, 68 and 83.
10. The method of claim 1 , wherein the at least one single nucleotide polymorphism is located in a mature miRNA portion of the microRNA region.
1 1. The method of claim 10, wherein the mature miRNA portion of the microRNA region comprises a sequence selected from the group consisting of SEQ ID NOs: 43, 67 and 82.
12. The method of any one of claims 1 - 1 1 , wherein the plant is maize.
13. The method of any one of claims 1 - 1 1 , wherein the plant trait is selected from the the group consisting of improved drought tolerance, improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
14. A method of identifying a plant having an improved trait, the method comprising:
(a) correlating at least one single nucleotide polymorphism in a microRNA region of a plant genome with the improved plant trait;
(b) sequencing a corresponding microRNA region in the plant; and
(c) detecting the at least one single nucleotide polymorphism in the microRNA region.
15. The method of claim 14, wherein the at least one single nucleotide polymorphism is located in a flanking sequence portion of the microRNA region.
16. The method of claim 15, wherein the flanking sequence portion of the microRNA region flanks a mature miRNA portion of the microRNA region comprising a sequence selected from the group consisting of SEQ ID NOs: 43, 67 and 82.
17. The method of claim 15, wherein the flanking sequence portion of the microRNA region flanks a pre-miRNA portion of the microRNA region comprising a sequence selected from the group consisting of SEQ ID NOs: 44, 68 and 83.
18. The method of claim 17, wherein the flanking sequence portion of the microRNA region is selected from the group consisting of
(a) nucleotides 1 - 490 of SEQ ID NO: 45;
(b) nucleotides 630 - 815 of SEQ ID NO: 45;
(c) nucleotides 1 - 273 of SEQ ID NO: 69;
(d) nucleotides 373 - 645 of SEQ ID NO: 69;
(e) nucleotides 1 - 303 of SEQ ID NO: 84; and
(f) nucleotides 429 - 779 of SEQ ID NO: 84.
19. The method of claim 18, wherein the at least one single nucleotide polymorphism is located at a nucleotide corresponding to a position selected from the group consisting of:
(a) position 216 of SEQ ID NO: 45, wherein the nucleotide is a C;
(b) position 301 of SEQ ID NO: 45, wherein the nucleotide is a C;
(c) position 743 of SEQ ID NO: 45, wherein the nucleotide is a T;
(d) position 444 of SEQ ID NO: 69, wherein the nucleotide is a C;
(e) position 500 of SEQ ID NO: 69, wherein the nucleotide is a C;
(f) position 120 of SEQ ID NO: 84, wherein the nucleotide is a C;
(g) position 218 of SEQ ID NO: 84, wherein the nucleotide is an A;
(h) position 575 of SEQ ID NO: 84, wherein the nucleotide is an A; and
(i) position 693 of SEQ ID NO: 84, wherein the nucleotide is an A.
20. The method of claim 14, wherein the at least one single nucleotide polymorphism is located in a pre-miRNA portion of the microRNA region.
21. The method of claim 20, wherein pre-miRNA portion of the microRNA region comprises a sequence selected from the group consisting of SEQ ID NOs: 43, 67 and 82.
22. The method of claim 20, wherein pre-miRNA portion of the microRNA region comprises a sequence selected from the group consisting of SEQ ID NOs: 44, 68 and 83.
23. The method of claim 14, wherein the at least one single nucleotide polymorphism is located in a mature miRNA portion of the microRNA region.
24. The method of claim 23, wherein the mature miRNA portion of the microRNA region comprises a sequence selected from the group consisting of SEQ ID NOs: 43, 67 and 82.
25. The method of any one of claims 14 - 24, wherein the plant is maize.
26. The method of any one of claims 14 - 24, wherein the plant trait is selected from the the group consisting of improved drought tolerance, improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
27. An isolated nucleic acid comprising a contiguous sequence of at least ten nucleotides of any one of:
(a) nucleotides 207 - 225 of SEQ ID NO: 45, wherein the nucleotide at a position corresponding to position 216 is a C;
(b) nucleotides 292 - 310 of SEQ ID NO: 45, wherein the nucleotide at a position corresponding to position 301 is a C;
(c) nucleotides 734 - 752 of SEQ ID NO: 45, wherein the nucleotide at a position corresponding to position 743 is a T;
(d) nucleotides 435 - 453 of SEQ ID NO: 69, wherein the nucleotide at a position corresponding to position 444 is a C;
(e) nucleotides 491 - 509 of SEQ ID NO: 69, wherein the nucleotide at a position corresponding to position 500 is a C;
(f) nucleotides 111 - 129 of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 120 is a C;
(g) nucleotides 209 - 227 of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 218 is an A;
(h) nucleotides 566 - 574 of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 575 is an A;
(i) nucleotides 684 - 702 of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 693 is an A; and
(j) a sequence complementary to any one of (a) - (i).
28. A method of producing a transgenic plant having an improved trait, the method comprising:
(a) expressing an isolated nucleic acid in a plant or plant part, the isolated nucleic acid selected from the group consisting of:
(i) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 43;
(ii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 44;
(iii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 45; (iv) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 45, wherein the nucleotide at a position corresponding to position 216 is a C; (v) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 45, wherein the nucleotide at a position corresponding to position 301 is a C;
(vi) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 45, wherein the nucleotide at a position corresponding to position 743 is a T;
(vii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 67;
(viii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 68;
(ix) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 69;
(x) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 69, wherein the nucleotide at a position corresponding to position 444 is a C;
(xi) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 69, wherein the nucleotide at a position corresponding to position 500 is a C;
(xii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 82;
(xiii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 83;
(xiv) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 84;
(xv) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 120 is a C;
(xvi) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 218 is an A;
(xvii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 575 is an A;
(xviii) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 84, wherein the nucleotide at a position corresponding to position 693 is an A; and
(xix) a sequence complementary to any one of (i) - (xviii); and (b) producing a transgenic plant having the improved strait thereby.
29. The method of claim 28, wherein the improved plant trait is selected from the the group consisting of improved ear height, improved plant height, improved grain yield at harvest moisture percentage, improved grain yield at standard moisture percentage, improved anthesis-silk interval, improved grain moisture adjusted percentage, improved grain moisture at harvest, reduced number of days to 50% plants pollen shedding, reduced number of days to 50% plants silking, improved yield grain adjustment at standard moisture, improved yield grain adjustment at harvest moisture, improved ratio of yield grain adjustment at standard moisture to grain moisture adjusted percentage, and improved ratio of yield grain adjustment at standard moisture to grain moisture at harvest.
30. The method of claim 28, further comprising:
(c) crossing the plant having the improved trait; and
(d) selecting at least one progeny plant having the improved trait.
31. The method of claim 30, further comprising the steps of:
(e) selfing the at least one progeny plant to produce at least one second generation progeny plant; and
(f) selecting a second generation progeny plant having the improved trait.
32. The method of claim 29, further comprising:
(c) crossing the plant having the improved trait; and
(d) selecting at least one progeny plant having the improved trait.
33. The method of claim 32, further comprising the steps of:
(e) selfing the at least one progeny plant to produce at least one second generation progeny plant; and
(f) selecting a second generation progeny plant having the improved trait.
34. A plant or plant part produced by the method of any one of claims 28 - 33.
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CN108504757A (en) * | 2017-04-14 | 2018-09-07 | 北京林业大学 | Probe into the method and system of regulatory mechanism between the gene and miRNAs that participate in forest miRNAs biologies formation access |
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CN102719543B (en) * | 2012-06-25 | 2013-12-04 | 中国科学院植物研究所 | Method for identifying plant varieties by utilizing chemical molecular formulas of nucleotides |
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CN110541046A (en) * | 2019-09-27 | 2019-12-06 | 中国农业科学院作物科学研究所 | Molecular marker related to corn kernel yield and quality |
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