Methods and compositions related to improved nitrogen use efficiency in tobacco

Abstract

The present disclosure provides metabolite signatures and genetic markers for tracking enhanced nitrogen utilization efficiency phenotypes in tobacco plants and infiltrating the enhanced nitrogen utilization efficiency phenotypes into tobacco plants. The present disclosure also provides tobacco plants comprising enhanced nitrogen utilization efficiency and methods for producing tobacco plants comprising enhanced nitrogen utilization efficiency. The present disclosure also provides recombinant polynucleotides and polypeptides for enhancing the nitrogen utilization efficiency of modified tobacco plants and tobacco plants comprising the provided recombinant polynucleotides and polypeptides.

Description

[0001] This application is a divisional application of the invention patent application with the application date of August 31, 2018, application number 2018800666507.3, and invention name “Methods and compositions related to improved nitrogen utilization efficiency in tobacco”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 553,501, filed September 1, 2017, which is incorporated herein by reference in its entirety.

[0004] Incorporation of Sequence Listing

[0005] This application contains a Sequence Listing filed electronically herein, comprising a 188,432-byte file entitled "P34523WO00_SL.txt" (available at and created on August 31, 2018, and is incorporated herein by reference in its entirety. Technical Field

[0006] The present disclosure provides compositions and methods for preparing and identifying tobacco plants comprising increased nitrogen use efficiency through breeding, transgenic pathways, and cisgene methods. Background Art

[0007] Fertilizer is a major cost for tobacco growers, and increased fertilizer use is associated with higher levels of alkaloids and tobacco-specific nitrosamines (TSNAs) in plant tissues. Different tobacco varieties require different levels of nitrogen fertilizer input to achieve maximum yield for each variety. For example, Maryland tobacco varieties typically require about 25% less nitrogen input to achieve maximum yield compared to Burley tobacco varieties.

[0008] Improving nitrogen use efficiency (NUE) in tobacco will increase the harvestable yield of tobacco per unit of input nitrogen fertilizer. Improvements in NUE also allow for lower farm input costs, reduced use and reliance on non-renewable energy sources required for nitrogen fertilizer production, and reduced environmental impacts of nitrogen fertilizer production and agricultural use.

[0009] Provided herein are methods and compositions for improving nitrogen utilization efficiency in tobacco. SUMMARY OF THE INVENTION

[0010] In one aspect, the present disclosure provides and includes a method of determining the NUE of a tobacco line, comprising obtaining at least one metabolite from a tobacco plant of the tobacco line, determining the amount of the metabolite obtained, and determining the NUE of the tobacco line based on the amount of the identified metabolite.

[0011] In one aspect, the present specification provides and includes a method for determining the NUE of a tobacco line using a metabolite signature, comprising isolating a metabolite signature from a tobacco plant of the tobacco line, determining the amount of each metabolite comprising the metabolite signature, and determining the NUE of the tobacco line by comparing the metabolite signature to a control metabolite signature from a control tobacco line comprising a known NUE.

[0012] In one aspect, the present specification provides and encompasses a method of breeding a tobacco line comprising a metabolite profile associated with enhanced NUE, comprising determining a metabolite profile of a first tobacco plant from a first tobacco line, wherein the first tobacco plant comprises enhanced NUE compared to a control tobacco plant lacking the metabolite profile, crossing the first tobacco plant with a second plant of a second tobacco line, and obtaining at least one progeny seed from the cross, wherein a progeny plant grown from the at least one progeny seed comprises the metabolite profile, and wherein the progeny plant comprises enhanced NUE compared to a control plant lacking the metabolite profile.

[0013] In one aspect, the present specification provides and encompasses a method for selecting a tobacco plant, comprising obtaining a population of tobacco plants, isolating at least one metabolite associated with enhanced NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant that comprises a higher amount of the at least one metabolite compared to a control tobacco plant. In a further aspect of the method, the selected tobacco plant comprises enhanced NUE compared to the control tobacco plant.

[0014] In one aspect, the present specification provides and encompasses a method of selecting a tobacco plant comprising obtaining a population of tobacco plants, isolating at least one metabolite associated with enhanced NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant comprising a lower amount of the at least one metabolite compared to a control tobacco plant.

[0015] In one aspect, the present specification provides and encompasses a method for screening a tobacco plant for a first metabolite signature associated with enhanced NUE, comprising isolating a first metabolite signature associated with enhanced NUE from a tobacco plant, determining the amount of at least one metabolite comprising the first metabolite signature, comparing the first metabolite signature to a second metabolite signature of a control tobacco plant comprising known NUE, and determining whether the first metabolite signature is associated with enhanced NUE.

[0016] In one aspect, the present specification provides and encompasses a modified tobacco seed, or a tobacco plant grown therefrom, comprising a cis-gene polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the modified tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis-gene polynucleotide when grown under the same conditions.

[0017] In one aspect, the present specification provides and encompasses a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0018] In one aspect, the present specification provides and encompasses a cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising a cis gene polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the modified tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0019] In one aspect, the present disclosure provides and encompasses a greenhouse, growth chamber, or field comprising the modified tobacco seeds or plants disclosed herein.

[0020] In one aspect, the present specification provides and encompasses a modified tobacco seed, or a tobacco plant grown therefrom, comprising at least one mutation in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant has enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking at least one mutation when grown under the same conditions.

[0021] In one aspect, the present disclosure provides and encompasses a recombinant DNA construct comprising a heterologous promoter operably linked to a guide RNA comprising at least 18 consecutive nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40.

[0022] In one aspect, the present specification provides and encompasses a cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising at least one mutation in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises enhanced NUE compared to an unmodified control tobacco plant lacking the at least one mutation when grown under the same conditions.

[0023] In one aspect, the present specification provides and encompasses a modified tobacco seed, or a tobacco plant grown therefrom, comprising a cis-gene polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA), said polynucleotide being at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises enhanced NUE compared to an unmodified control tobacco plant lacking the cis-gene polynucleotide when grown under the same conditions.

[0024] In one aspect, the present specification provides and encompasses a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0025] In one aspect, the present specification provides and includes a cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising a cis-gene polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding an sRNA at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, and wherein the modified tobacco seed or tobacco plant comprises enhanced NUE compared to an unmodified control tobacco plant lacking the cis-gene polynucleotide when grown under the same conditions.

[0026] In one aspect, the present specification provides and encompasses a method for enhancing NUE in a tobacco plant, comprising introducing a cis-gene nucleic acid molecule into a tobacco cell, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the cis-gene nucleic acid molecule.

[0027] In one aspect, the present specification provides and encompasses a method for enhancing NUE in a tobacco plant, comprising introducing into a tobacco cell a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41-56, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the modification.

[0028] In one aspect, the present specification provides and encompasses a method for enhancing NUE in a tobacco plant, comprising introducing into a tobacco cell a nucleic acid encoding a small RNA (sRNA) homologous to at least 18 contiguous nucleic acids of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of SEQ ID NOs: 41-56, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the sRNA.

[0029] In one aspect, the present specification provides and includes a method comprising providing a first population of tobacco plants comprising enhanced NUE, genotyping the first population of tobacco plants for the presence of a molecular marker within 20 cM of the enhanced NUE locus; and selecting one or more tobacco plants that were genotyped and found to contain the molecular marker.

[0030] In one aspect, the present specification provides and includes a method comprising providing a first population of tobacco plants, genotyping the first population of tobacco plants for the presence of an enhanced NUE allele at a locus encoded by a sequence selected from the group consisting of SEQ ID NOs: 9-16; and selecting one or more genotyped tobacco plants comprising the enhanced NUE allele.

[0031] In one aspect, the present specification provides and includes a method for introgressing an enhanced NUE trait into a tobacco variety, comprising crossing a first tobacco variety comprising an enhanced nitrogen use efficiency trait with a second tobacco variety lacking the enhanced nitrogen use efficiency trait, obtaining progeny seeds from the cross, genotyping at least one progeny seed for a molecular marker associated with the enhanced nitrogen use efficiency trait, wherein the molecular marker is located within 20 cM of a locus having a sequence selected from the group consisting of SEQ ID NOs: 9-16, and selecting progeny seeds comprising the enhanced nitrogen use efficiency trait.

[0032] In one aspect, the present specification provides and encompasses a method for selecting tobacco plants having an enhanced NUE trait, comprising isolating nucleic acid from a tobacco germplasm collection, assaying the isolated nucleic acid for one or more markers located within 20 cM of a locus selected from the group consisting of SEQ ID NOs: 9-16, and selecting tobacco plants comprising the enhanced NUE trait.

[0033] In one aspect, the present specification provides and encompasses a method for selecting tobacco plants having an enhanced NUE trait, comprising isolating nucleic acid from a tobacco germplasm collection, assaying the isolated nucleic acid for one or more markers within 20 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64, and selecting tobacco plants comprising the enhanced NUE trait.

[0034] Brief description of sequence

[0035] SEQ ID NOs: 1-8 are the amino acid sequences of genes positively correlated with enhanced NUE in root tissue, leaf tissue, or both.

[0036] SEQ ID NOs: 9-16 are nucleotide sequences of genes positively correlated with enhanced NUE in root tissue, leaf tissue, or both.

[0037] SEQ ID NOs: 17-19 are nucleotide sequences of promoter regions of genes having leaf-preferential expression.

[0038] SEQ ID NOs: 20-24 are nucleotide sequences of promoter regions of genes having root-preferred expression.

[0039] SEQ ID NOs: 25-40 are the amino acid sequences of genes negatively correlated with enhanced NUE in root tissue, leaf tissue, or both.

[0040] SEQ ID NOs: 41-56 are nucleotide sequences of genes negatively correlated with enhanced NUE in root tissue, leaf tissue, or both.

[0041] SEQ ID NOs: 57-64 are nucleotide sequences of SNP markers comprising polymorphisms associated with enhanced NUE.

[0042] SEQ ID NO: 65 is the backbone sequence of the expression vector p45-2-7. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Four gene clusters associated with NUE in the tobacco genome are depicted. Genes differentially expressed under low nitrogen and normal nitrogen conditions in plants with NUE metabolite fingerprints (related genes) are shown. The total number of differentially expressed genes (DEGs) is also shown, regardless of the NUE metabolic fingerprint.

[0044] Figure 2 Depicted is a 2-megabase region of tobacco chromosome 11 covered by superscaffold 1. Superscaffold 1 is a contig of scaffolds A and B. Of the 79 expressed genes located in this region, 56 genes were differentially expressed.

[0045] Figure 3 Depicted are the allelic composition of 23 Burley and 6 Maryland varieties associated with NUE on tobacco chromosome 11. Lines 1, 2, and 3 are Burley lines containing the favorable Maryland allele at SEQ ID NO:58, and line 4 is a standard Burley line containing the unfavorable Burley allele at SEQ ID NO:58.

[0046] Figure 4 Describes how Figure 3 Chlorophyll loss, growth and yield of lines 1-4 and the MD609 control (C) are shown. Lines 1, 2 and 3 are burley lines containing the favorable Maryland allele at SEQ ID NO: 58, and line 4 is a standard burley line containing the unfavorable burley allele at SEQ ID NO: 58.

[0047] Figure 5 Depicted are the yield (grams fresh weight per plant) of greenhouse-grown T1 plants overexpressing genes positively correlated with yield increase under nitrogen stress. Means and standard deviations based on 9 plants per sample are shown.

[0048] Figure 6 Yield (lbs / acre) of two independent field-grown F4 lines (NUE-2 and NUE-3) after harvest is depicted. Test lines were generated from a cross between MD609 and Burley TN90 as described. Means and standard deviations compared to control TN90 Burley tobacco are provided. Detailed Description of the Invention

[0050] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. Those skilled in the art will recognize that many methods can be used in the practice of the present disclosure. In fact, the present disclosure is in no way limited to the methods and materials described. For the purposes of this disclosure, the following terms are defined as follows.

[0051] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. When a term is provided in the singular, the inventors also contemplate the aspects of the disclosure described by the plural form of that term. When there are differences in the terms and definitions used in the references incorporated by reference, the terms used in this application shall have the definitions given therein. Other technical terms used have their ordinary meaning in the field in which they are used, as exemplified in various field-specific dictionaries, such as "The American Science Dictionary" (Editors of the American Heritage Dictionary, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to the mechanism of action or mode of action. References thereto are provided for illustrative purposes only.

[0052] Unless otherwise indicated, the practice of the present disclosure involves conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, biotechnology, metabolomics, plant breeding, and genetics, which are within the skill of the art. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)); theseries Methods In Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJMacPherson, BDHames and GRTaylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (RI Freshney, ed. (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; CN Stewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) Plant Transformation Technologies (Wiley-Blackwell); and RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0053] Any references cited herein (eg, all patents, published patent applications, and non-patent publications) are hereby incorporated by reference in their entirety.

[0054] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0055] As used herein, in the context of two polynucleotides or polypeptide sequences, the term "sequence identity" or "consistency" refers to the residues in the two sequences being identical when the maximum correspondence is aligned over a specified comparison window. When using a percentage of sequence identity for a protein, it is recognized that non-identical residue positions are typically different by conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues with similar chemical properties (such as charge or hydrophobicity), and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upwards to correct the conservative properties of the substitutions. Sequences that differ by such conservative substitutions are referred to as having "sequence similarity" or "similarity." Any suitable computer program can be used to compare two or more sequences. For example, a widely used and recognized computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).

[0056] As used herein, the term "complementary" with respect to nucleic acid molecules refers to the pairing of nucleotide bases such that adenine is complementary to thymine or uracil, and guanine is complementary to cytosine. Two complementary nucleic acid molecules are capable of hybridizing to each other. For example, the two strands of double-stranded DNA are complementary to each other.

[0057] A specific polynucleotide having a length of at least three nucleotides can be referred to as an "oligonucleotide." Nucleic acid molecules provided herein include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and functional analogs thereof, such as complementary DNA (cDNA). Nucleic acid molecules provided herein can be single-stranded or double-stranded. Nucleic acid molecules comprise the nucleotide bases adenine (A), guanine (G), thymine (T), and cytosine (C). Uracil (U) replaces thymine in RNA molecules. The symbol "R" can be used to represent a purine (e.g., A or G) nucleotide base. The symbol "Y" can be used to represent a pyrimidine (e.g., C or T) nucleotide base. The symbol "W" can be used to represent an A or T nucleotide base. The symbol S can be used to represent a G or C nucleotide base. The symbol "M" can be used to represent an A or C nucleotide base. The symbol "K" can be used to represent a G or T nucleotide base. The symbol "B" can be used to represent a G, C, or T nucleotide base. The symbol "H" can be used to represent an A, C, or T nucleotide base. The symbol "D" can be used to represent an A, G, or T nucleotide base. The symbol "V" can be used to represent an A, G, or C nucleotide base. The symbol "N" can be used to represent any nucleotide base (e.g., A, G, C, T, or U).

[0058] The use of the term "polynucleotide" is not intended to limit the present disclosure to polynucleotides comprising DNA. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like.

[0059] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein.

[0060] Nucleic acid molecules, polypeptide or protein provided herein can be isolated or substantially purified." isolated " or " purified " nucleic acid molecules, polypeptide, protein or its biologically active part are in fact or substantially free of the polynucleotide or protein companion or interactional component usually found in its naturally occurring environment. For example, when produced by recombinant technology, isolated or purified polynucleotide or protein are substantially free of other cellular materials or culture medium, or when chemosynthesis, are substantially free of chemical precursors or other chemicals. On the one hand, the isolated polynucleotide provided herein can comprise and be less than 10000 nucleotide, less than 5000 nucleotide, less than 4000 nucleotide, less than 3000 nucleotide, less than 2000 nucleotide, less than 1000 nucleotide, less than 500 nucleotide or less than the nucleotide sequence of 100 nucleotide, and described nucleotide sequence is positioned at the polynucleotide flank in the genomic DNA of the cell from which polynucleotide is derived naturally. In one aspect, the isolated polynucleotides provided herein can comprise a nucleic acid sequence of 100-10,000 nucleotides, 500-10,000 nucleotides, 1,000-10,000 nucleotides, 2,000-10,000 nucleotides, 3,000-10,000 nucleotides, 4,000-10,000 nucleotides, 1-500 nucleotides, 1-1,000 nucleotides, 1-2,000 nucleotides, 1-3,000 nucleotides, 1-4,000 nucleotides, 1-5,000 nucleotides, 1-10,000 nucleotides, 100-500 nucleotides, 100-1,000 nucleotides, 100-2,000 nucleotides, 100-3,000 nucleotides, or 100-4,000 nucleotides that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. On the other hand, the isolated polypeptide provided herein is substantially free of cellular material in the preparation, and the content of the chemical precursors or non-target protein chemicals of the preparation is less than 30%, less than 20%, less than 10%, less than 5% or less than 1% (dry weight). The fragments of the disclosed polynucleotides and the polypeptides encoded therefrom are also encompassed in the present invention. The polynucleotide fragments can encode polypeptide fragments that retain the biological activity of the native polypeptide. Alternatively, the polynucleotide fragments used as hybridization probes or PCR primers using methods known in the art do not typically encode fragment polypeptides that retain biological activity. Depending on the desired result, the polynucleotide fragments provided herein can be at least 20 nucleotides, at least 50 nucleotides, at least 70 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, and up to the full-length polynucleotides encoding the polypeptide of the present invention.

[0061] The routine techniques of this area can be used to separate nucleic acids. For example, any method can be used to separate nucleic acids, including but not limited to recombinant nucleic acid technology and / or polymerase chain reaction (PCR). For example, common PCR technology is described in PCR Primer: Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. For example, recombinant nucleic acid technology includes restriction enzyme digestion and connection, which can be used to separate nucleic acids. Isolated nucleic acids can also be chemically synthesized as single nucleic acid molecules or as a series of oligonucleotides. Polypeptides can be purified from natural sources (such as biological samples) by known methods (such as DEAE ion exchange, gel filtration and hydroxyapatite chromatography). For example, polypeptides can also be purified by expressing nucleic acids in expression vectors. In addition, purified polypeptides can be obtained by chemical synthesis. The purity of the polypeptide can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

[0062] In one aspect, the present disclosure provides methods for detecting recombinant nucleic acids and polypeptides in plant cells. Without limitation, nucleic acids can also be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0063] Antibodies can be used to detect polypeptides. Techniques for using antibodies to detect polypeptides include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Antibodies provided herein can be polyclonal or monoclonal antibodies. Antibodies having specific binding affinity for the polypeptides provided herein can be prepared using methods well known in the art. Antibodies provided herein can be attached to solid supports, such as microtiter plates, using methods well known in the art.

[0064] Detection can be performed using a detectable label (e.g., an amplification product, a hybridization complex, a polypeptide). The term "label" is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0065] As used herein, the phrase "associated with" or "associated with" refers to an identifiable and / or measurable relationship between two entities. For example, the phrase "associated with enhanced NUE" refers to a trait, locus, gene, allele, marker, phenotype, etc., or expression thereof, the presence or absence of which can affect the extent, degree, and / or rate of a plant or part of interest having an enhanced NUE trait. Thus, a marker is "associated with a trait" when it is associated with the trait, and when the presence of the marker is an indicator of whether and / or to what extent the desired trait or trait form will occur in a plant / germplasm containing the marker. Similarly, a marker is "associated with an allele" when it is associated with the allele, and when the presence of the marker is an indicator of whether the allele is present in a plant / germplasm containing the marker. For example, a "marker associated with an enhanced NUE allele" refers to a marker whose presence or absence can be used to predict whether and to what extent a plant will display an enhanced NUE phenotype.

[0066] As used herein, "centimorgan" (cM) is a unit of measure for recombination frequency and genetic distance between two loci. One cM is equal to a 1% chance that a marker at one locus will segregate with a marker at a second locus due to hybridization in one generation.

[0067] As used herein, "closely linked" means that a marker or locus is within about 20 cM, 15 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM of another marker or locus. For example, 20 cM means that recombination occurs between the marker and the locus at a frequency of about 20% or less.

[0068] As used herein, "plant" refers to the entire plant. Cells or tissue cultures derived from plants can include any plant component or plant organ (e.g., leaves, stems, roots, etc.), plant tissue, seeds, plant cells, and / or their progeny. Progeny plants can be from any progeny generation, such as F1, F2, F3, F4, F5, F6, F7, etc. A plant cell is a biological cell of a plant, taken from a plant or derived by culturing a cell taken from a plant.

[0069] As used herein, the tobacco plant can be from any plant of the genus Nicotiana tabacum, including but not limited to Nicotiana tabacum tabacum; Nicotiana tabacum amplexicaulis, PI 271989; Nicotiana tabacum benthamiana PI 555478; Nicotiana tabacum bigelovii PI 555485; Nicotiana tabacum debneyi; Nicotiana tabacum excelsior PI 224063; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum goodspeedii PI 241012; Nicotiana tabacum gossei PI 555478; Nicotiana tabacum glutinosa PI 555476; Nicotiana tabacum glutinosa PI 555477; Nicotiana tabacum glutinosa ...7; Nicotiana tabacum glutinosa PI 555477; Nicotiana tabacum glutinosa PI 555477; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum goodspeedii PI 241012; Nicotiana tabacum gossei PI 555477; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum glutinosa PI 555507; Nicotiana tabacum glutinosa PI 555507; Nico 230953; Nicotiana tabacum hesperis (PI 271991); Nicotiana tabacum knightiana (PI 555527); Nicotiana tabacum maritima (PI 555535); Nicotiana tabacum megalosiphon (PI 555536); Nicotiana tabacum nudicaulis (PI 555540); Nicotiana tabacum paniculata (PI 555545); Nicotiana tabacum plumbaginifolia (PI 555548); Nicotiana tabacum repanda (PI 555552); Nicotiana tabacum rustica (PI); Nicotiana tabacum suaveolens (PI) 230960; Nicotiana tabacum sylvestris PI 555569; Nicotiana tabacum tomentosa PI 266379; Nicotiana tabacumtomentosiformis;and Nicotiana tabacum trigonophylla PI 555572. ;

[0070] On the one hand, plant component provided herein includes but not limited to leaf, stem, root, seed, flower, pollen, anther, ovule, pedicel, fruit, meristem, cotyledon, hypocotyl, pod, embryo, endosperm, explant, callus, tissue culture, tender shoot, cell and protoplast.In other respects, present disclosure provides and is not the tobacco plant cell, tissue and the organ of the natural propagation of propagation material and mediation plant.On the other hand, present disclosure also provides and is the tobacco plant cell, tissue and the organ of the natural propagation of propagation material and mediation plant.On the other hand, present disclosure provides and can not keep self tobacco plant cell, tissue and the organ through photosynthesis.On the other hand, present disclosure provides somatic tobacco plant cell.Contrary to reproductive cell, somatic cell does not mediate plant propagation.

[0071] The cell, tissue and organ provided can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryo, endosperm, root, tender shoot, stem, pod, flower, inflorescence, petiole, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem and vascular tissue.On the other hand, the disclosure provides tobacco plant chloroplast.In further aspect, the disclosure provides epidermal cell, stomatal cell, leaf hair (trichome), root hair or storage root.On the other hand, the disclosure provides tobacco protoplast.

[0072] Technician understands that tobacco plant is through seed natural propagation, rather than through asexual propagation or vegetative propagation.On the one hand, the disclosure provides tobacco endosperm.On the other hand, the disclosure provides tobacco endosperm cell.In further aspect, the disclosure provides male or female sterile tobacco plant, it can not breed under the situation that does not have human intervention.

[0073] In one aspect, the present disclosure provides methods and compositions related to modified tobacco plants, seeds, plant components, plant cells, and products made from modified tobacco plants, seeds, plant parts, and plant cells. In one aspect, the modified seeds provided herein produce modified plants provided herein. In one aspect, the modified plants, seeds, plant components, plant cells, or plant genomes provided herein comprise recombinant DNA constructs provided herein. In another aspect, the cured tobacco materials or tobacco products provided herein comprise the modified tobacco plants, plant components, plant cells, or plant genomes provided herein.

[0074] As used herein, "modified" refers to plants, seeds, plant components, plant cells, and plant genomes that have undergone mutagenesis, genome editing, genetic transformation, or a combination thereof.

[0075] As used herein, " isogenic transgene " or " cisgene " refer to the genetic modification of plant, plant cell or plant genome, wherein all components (for example, promoter, donor nucleic acid, selection gene) only have plant origin (that is, do not use components of non-plant origin).On the one hand, modified plant, plant cell or plant genome provided herein are cisgene.Cigene plant, plant cell and plant genome provided herein can produce ready-to-use tobacco strain.On the other hand, modified tobacco plant provided herein does not comprise the genetic material or sequence of non-tobacco.

[0076] As used herein, "functional fragment" or "functional fragment thereof" refers to a nucleotide or amino acid sequence of any size that retains the function of the full-length sequence to which it refers. In one aspect, the length of a functional fragment can be at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000 or more than 5000 nucleotides. In one aspect, the length of the functional fragment can be at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000 or more than 2000 amino acids. In one aspect, the length of the functional fragment can be 5-5000 nucleotides, 10-4000 nucleotides, 25-3000 nucleotides, 50-2000 nucleotides, 75-1000 nucleotides, 100-900 nucleotides, 150-800 nucleotides, 200-700 nucleotides, 250-600 nucleotides or 300-500 nucleotides. In one aspect, the length of the functional fragment can be 5-2000 amino acids, 10-1000 amino acids, 25-900 amino acids, 50-800 amino acids, 50-800 amino acids, 75-700 amino acids, 100-600 amino acids, 150-500 amino acids, 200-400 amino acids or 250-300 amino acids. In a further aspect, the polynucleotides described herein are expected in their entirety as well as any functional fragment thereof. In a further aspect, the polypeptides described herein are expected in their entirety as well as any functional fragment thereof. In a further aspect, the polynucleotides with the sequences of SEQ ID NOs: 9-24 and 41-56 are expected in their entirety as well as any functional fragment thereof. In a further aspect, the polypeptides with the sequences of SEQ ID NOs: 1-8 and 25-40 are expected in their entirety as well as any functional fragment thereof.

[0077] As used herein, the term "nitrogen use efficiency" (NUE) refers to the ability of a plant to absorb, assimilate and / or use nitrogen (e.g., from soil, water and / or nitrogen fertilizer). NUE genes affect yield and have the effect of improving nitrogen utilization in crop plants. Enhanced nitrogen use efficiency can be attributed to increased uptake and assimilation of nitrogen fertilizer and / or subsequent remobilization and reuse of accumulated nitrogen reserves, as well as increased tolerance of the plant to stress conditions (e.g., low nitrogen environments). NUE genes can be used to alter the genetic makeup of plants to increase productivity under current fertilizer application standards or to maintain productivity under significantly reduced fertilizer or reduced nitrogen use efficiency.

[0078] NUE has been defined in various ways, but yield per unit of available nitrogen in the soil integrates all the key parameters used to assess the adaptability of crop cultivars and is a commonly used measure of NUE. For example, see Ladha et al., 2005. Advances in Agronomy, 87:85-156, which is incorporated herein in its entirety. This metric is sometimes referred to as "agricultural NUE." As another measure of NUE, the ratio of plant product (e.g., tobacco leaf tissue) to above-ground nitrogen in the plant can be determined (sometimes referred to as "physiological NUE"). Enhanced NUE is associated with three key components: 1) no significant difference in yield when grown at 25% normal nitrogen compared to plants grown at 100% normal nitrogen; 2) a reduced rate of chlorophyll loss compared to plants without enhanced NUE; and 3) no significant difference in mature leaf mass when grown at 25% normal nitrogen compared to plants grown at 100% normal nitrogen. In a preferred aspect, plants having enhanced NUE are capable of producing similar yield and leaf quality when grown at a 25% burley fertilizer rate compared to burley plants grown at a 100% normal burley fertilizer rate.

[0079] At least five pathways and indicators of NUE are used in the art and are discussed below.

[0080] (1) Partial factor productivity (PFP) from applied nitrogen (N) is a measure of how much yield is produced per unit of nitrogen applied:

[0081] PFP N = Yield (kg) / N applied (kg)

[0082] PFP N =Y +N / FN

[0083] where Y +N Y is the yield (kg / ha) and FN is the fertilizer rate (kg / ha).

[0084] (2) Agronomic efficiency (AE) of applied nitrogen (N) is a measure of how much additional yield is produced per unit of nitrogen applied:

[0085] AE N = Yield increase (kg) / N applied (kg)

[0086] AE N =(Y +N -Y 0N ) / FN

[0087] where Y +N is the yield in the N treatment (kg / ha); Y 0N is the yield of the control treatment without N application (kg / ha); and FN is the amount of N fertilizer applied (kg / ha).

[0088] (3) Applied nitrogen (N) recovery efficiency (RE) is a measure of how much of the applied nitrogen is recovered and taken up by crops.

[0089] RE N = N absorbed (kg) / N applied (kg)

[0090] RE N =(UN +N -UN 0N ) / FN

[0091] Among them, UN +N is the total plant N uptake (kg / ha) measured in aboveground biomass at physiological maturity in plots receiving the applied proportional FN (kg / ha); and UN 0N is the total N uptake in the control plot without N addition.

[0092] (4) Physiological efficiency (PE) of applied nitrogen (N) is a measure of how much additional yield is produced per additional unit of nitrogen uptake.

[0093] PE N = Yield increase (kg) / Fertilizer N absorbed (kg)

[0094] PE N =(Y +N -Y 0N ) / (UN +N -UN 0N )

[0095] where Y +N is the yield in the N treatment (kg / ha); Y 0N is the yield of the control treatment without N application (kg / ha); UN +Nis the total N uptake (kg / ha) in the treatment receiving the applied fertilizer N; and UN 0N is the total N uptake (kg / ha) in the treatment without N fertilizer application.

[0096] (5) Internal efficiency (IE) of nitrogen (N) relates to how much yield is produced per unit of N absorbed from fertilizers and inherent (e.g. soil) nutrient sources:

[0097] IE N = production (kg) / N absorbed (kg)

[0098] IE N =Y / UN

[0099] where Y is yield (kg / ha); and UN is total N uptake (kg / ha).

[0100] Nitrogen can be in any form, including organic and / or inorganic forms. Forms of nitrogen include, but are not limited to, nitrates (e.g., ammonium nitrate, calcium nitrate, potassium nitrate), nitrites, ammonia, ammoniacal liquor, anhydrous ammonia, ammonium sulfate, diammonium phosphate, low-pressure nitrogen solution, pressureless nitrogen solution, urea, and urea-ammonium nitrate (UAN). In one aspect, the nitrogen is in a form that is immediately available to plants (e.g., ammonia and / or nitrate) and / or can be easily converted into a form that is available to plants (e.g., urea).

[0101] In one aspect, the modified tobacco plants provided herein that contain enhanced NUE contain increased nitrogen uptake compared to control tobacco plants. In another aspect, the modified tobacco plants provided herein that contain enhanced NUE contain increased nitrogen assimilation compared to control tobacco plants. In a further aspect, the modified tobacco plants provided herein that contain enhanced NUE contain increased yield compared to control tobacco plants. In yet another aspect, the modified tobacco plants provided herein that contain enhanced NUE contain increased yield under low nitrogen conditions compared to control tobacco plants. In a preferred aspect, the low nitrogen conditions used in the field are about 25% nitrogen compared to the levels typically used by those skilled in the art. In another aspect, the low nitrogen conditions used in the field are about 5%-50% nitrogen compared to the levels typically used by those skilled in the art. In a greenhouse environment, low nitrogen conditions are about 25 parts per million (ppm), and normal nitrogen conditions are about 100 ppm. In another aspect, the low nitrogen conditions used in the greenhouse can be 5 ppm-50 ppm.

[0102] In one aspect, the modified tobacco plants provided herein comprising enhanced NUE comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500% increase in yield compared to control tobacco plants grown under similar growth conditions. In one aspect, the modified tobacco plants provided herein comprising enhanced NUE comprise a 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 10-200%, 10-300%, 10-400%, 10-500%, or a 5-500% increase in yield compared to a control tobacco plant grown under similar growth conditions.

[0103] In one aspect, a population of modified tobacco plants provided herein comprising enhanced NUE comprises an increase in yield of at least 0.25 kg / ha, at least 0.5 kg / ha, at least 0.75 kg / ha, at least 1 kg / ha, at least 2 kg / ha, at least 3 kg / ha, at least 4 kg / ha, at least 5 kg / ha, at least 6 kg / ha, at least 7 kg / ha, at least 8 kg / ha, at least 9 kg / ha, at least 10 kg / ha, at least 15 kg / ha, at least 20 kg / ha, at least 25 kg / ha, at least 30 kg / ha, at least 35 kg / ha, at least 40 kg / ha, at least 45 kg / ha, at least 50 kg / ha, at least 75 kg / ha, at least 100 kg / ha, at least 200 kg / ha, at least 300 kg / ha, at least 400 kg / ha, or at least 500 kg / ha, as compared to a population of control tobacco plants grown under similar growing conditions. In another aspect, the modified tobacco plant population provided herein comprising enhanced NUE comprises 0.25 kg / ha-100 kg / ha, 0.5 kg / ha-100 kg / ha, 0.75 kg / ha-100 kg / ha, 1 kg / ha-100 kg / ha, 2 kg / ha-100 kg / ha, 3 kg / ha-100 kg / ha, 4 kg / ha-100 kg / ha, 5 kg / ha-100 kg / ha, 6 kg / ha-100 kg / ha, 7 kg / ha-100 kg / ha, 8 kg / ha-100 kg / ha, 9 kg / ha-100 kg / ha, 10 kg / ha-100 kg / ha, 15 kg / ha-100 kg / ha, 16 kg / ha-100 kg / ha, 17 kg / ha-100 kg / ha, 18 kg / ha-100 kg / ha, 19 kg / ha-100 kg / ha, 20 kg / ha-100 kg / ha, 21 kg / ha-100 kg / ha, 22 kg / ha-100 kg / ha, 23 kg / ha-100 kg / ha, 24 kg / ha-100 kg / ha, 25 kg / ha-100 kg / ha, 26 kg / ha-100 kg / ha, 27 kg / ha-100 kg / ha, 28 kg / ha-100 kg / ha, 29 kg / ha-100 kg / ha, 30 kg / ha-100 kg / ha, 31 kg / ha-100 kg / ha, kg / ha-100kg / ha, 9kg / ha-100kg / ha, 10kg / ha-100kg / ha, 15kg / ha-100kg / ha, 20kg / ha-100kg / ha, 30kg / ha-100kg / ha, 40kg / ha-100kg / ha, 50kg / ha-100kg / ha, 75kg / ha-100kg / ha, 100kg / ha-500kg / ha, 100kg / ha-400kg / ha, 100-300kg / ha or 100kg / ha-200kg / ha. As used herein, tobacco plant " colony " can have any size, for example 5,10,15,20,25,30,35,40,50,100,500,1000,5000,10000,25000,50000,100000,500000 or more.Colony can be from single variety, cultivar or strain.Can use any breeding technique known in the art to produce colony.

[0104] In one aspect, the modified tobacco plants provided herein comprising enhanced NUE comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 more leaves than a control tobacco plant grown under similar growth conditions. In another aspect, the modified tobacco plants provided herein comprising enhanced NUE comprise 1-25, 2-25, 3-25, 4-25, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 11-25, 12-25, 13-25, 14-25, 15-25, or 20-25 more leaves than a control tobacco plant grown under similar growth conditions.

[0105] As used herein, "comparable conditions", "similar conditions" or "similar growing conditions" refer to similar environmental conditions, agronomic practices and / or curing methods for growing or curing tobacco and making meaningful comparisons between two or more plant genotypes, such that neither environmental conditions nor agronomic practices (including curing methods) contribute to or explain any differences observed between two or more plant genotypes. For example, environmental conditions include light, temperature, water, humidity and nutrition (e.g., nitrogen and phosphorus). For example, agronomic practices include sowing, pruning, undercutting, transplanting, topping, bifurcating and curing. See Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), Chapter 4B and Chapter 4C of pp.70-103.

[0106] On the one hand, the modified plant, seed, plant part or plant cell provided herein comprise one or more non-naturally occurring mutations.On the one hand, the mutation provided herein improves nitrogen use efficiency.For example, the mutation type provided herein includes substitution (point mutation), disappearance, insertion, repetition and inversion.It is expected that this mutation is present in the coding region of the gene; However, it is also expected that the mutation in the untranslated region of the promoter or other regulatory regions, introns, intron-exon boundaries or genes is present.

[0107] On the one hand, method provided herein can produce the tobacco plant with enhanced nitrogen utilization efficiency compared with control tobacco plant.Mutagenesis method includes but is not limited to chemical mutagenesis, for example, with ethyl methyl sulfate (EMS) process seed (Hildering and Verkerk, In, The use of induced mutations in plantbreeding.Pergamon Press, pp.317-320,1965); Or UV radiation, X-ray, electron beam, ion beam (for example carbon ion beam, helium ion beam, neon ion beam) and fast neutron irradiation (for example, referring to Verkerk, Neth.J.Agric.Sci.19:197-203,1971; Poehlman, Breeding Field Crops, V Nostrand Reinhold, New York (3.sup.rd ed.), 1987; and Tanaka, J. Radiat. Res. 51:223-233, 2010); transposon markers (Fedoroff et al., 1984; U.S. Pat. No. 4,732,856 and U.S. Pat. No. 5,013,658) and T-DNA insertion methods (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis involves chemically inducing random point mutations across the genome length. Fast neutron mutagenesis involves exposing seeds to neutron bombardment, which causes large deletions through double-stranded DNA breaks. Transposon markers involve inserting a transposon into an endogenous gene to reduce or eliminate gene expression.

[0108] In addition, rapid and automatable methods for screening chemically induced mutations, TILLING (targeted induced localized lesions of the genome), selective endonuclease digestion using denaturing high performance liquid chromatography (HPLC) or selected PCR products are also suitable for use in the present disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18: 455-457. Mutations that affect gene expression or interfere with gene function as provided herein can be determined using methods well known in the art. Insertion mutations in gene exons typically result in null mutants. Mutations in conserved residues can be particularly effective in inhibiting protein function.

[0109] Can be by any method screening and selection mutagenic tobacco plant known to those of ordinary skill in the art.The example of screening and selection method includes but not limited to Southern blotting, PCR amplification for detecting polynucleotide, Northern blotting, RNase protection, primer extension, RT-PCR amplification for detecting rna transcript, Sanger order-checking, next generation sequencing technology (for example, Illumina, PacBio, Ion Torrent, 454), the enzymatic analysis for detecting the enzyme of polypeptide and polynucleotide or ribozyme activity, and protein gel electrophoresis, Western blotting, immunoprecipitation and enzyme-linked immunosorbent assay for detecting polypeptide.Other technologies (such as in situ hybridization, enzyme staining and immunostaining) also can be used for detecting the existence or expression of polypeptide and / or polynucleotide.The method for carrying out all indicated technologies is known in the art.

[0110] On the one hand, the plant genome provided herein is mutated (edited) by a nuclease selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR / Cas9 nuclease, a CRISPR / Cpf1 or a CRISPR / Cmx1 nuclease. On the other hand, the plant genome provided herein is mutated by a CRISPR / CasX or CRISPR / CasY nuclease. As used herein, "editing" or "genome editing" refers to the directed mutagenesis of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 nucleotides of an endogenous plant genome nucleic acid sequence, or removes or replaces the nucleic acid sequence of an endogenous plant genome.

[0111] This paper also provides and uses any suitable transformation method known in the art to transform tobacco plant with recombinant construct as herein described or expression cassette.The method that polynucleotide sequence is introduced into tobacco plant is known in the art, includes but not limited to stable transformation method, transient transformation method and virus-mediated method." stable transformation" refers to that the target nucleotide construct of wherein introducing plant is integrated in the genome of vegetable cell and can be inherited by its offspring. " transient transformation " is intended to represent that sequence is introduced into plant or vegetable cell, and only in plant or vegetable cell transient expression or only transiently is present in plant or vegetable cell.

[0112] On the one hand, method and composition provided herein include one or more polynucleotides are introduced into one or more plant cells.On the one hand, modification plant genome provided herein is to include polynucleotides or recombinant DNA constructs introduced.As used herein, "plant genome" refers to the nuclear genome, mitochondrial genome or plastid (e.g., chloroplast) genome of plant cells.On the other hand, polynucleotides provided herein are integrated into artificial chromosomes.On the one hand, artificial chromosomes comprising polynucleotides provided herein are integrated into plant cells.

[0113] On the one hand, the modified plant, seed, plant component, vegetable cell or plant genome provided herein comprise one or more transgenes.On the one hand, the transgenic improvement nitrogen utilization efficiency in the tobacco plant provided herein.As used herein, " transgenic " refers to the polynucleotide transferred in the genome by any method known in the art.On the one hand, transgenic is an exogenous polynucleotide.On the one hand, transgenic is an endogenous polynucleotide, which is integrated into its non-existent new genome site usually.Therefore, in appropriate cases, transgenic also can be a cisgene.

[0114] On the one hand, transgenic provided herein comprises recombinant DNA construct.On the one hand, recombinant DNA construct provided herein or expression cassette can comprise the selective marker gene for selecting transgenic cells.Selective marker gene includes but is not limited to the gene encoding antibiotic resistance, such as the gene encoding neomycin phosphotransferase II (NPTII) and hygromycin phosphotransferase (HPT), and the gene imparting resistance to herbicides, such as glufosinate, bromoxynil, imidazolinones, triazolopyrimidines, sulfonylureas (such as chlorsulfuron and sulfometuron-methyl) and 2,4-dichlorophenoxyacetic acid ester (2,4-D).Other selective markers include phenotypic markers, such as beta-galactosidase and fluorescent protein, such as green fluorescent protein (GFP).

[0115] In one aspect, the methods and compositions provided herein comprise vectors. As used herein, the terms "vector" or "plasmid" are used interchangeably and refer to circular double-stranded DNA molecules that are physically separated from chromosomal DNA. In one aspect, the plasmid or vector used herein is capable of replicating in vivo. As used herein, a "transformation vector" is a plasmid capable of transforming plant cells. In one aspect, the plasmid provided herein is a bacterial plasmid. In another aspect, the plasmid provided herein is an Agrobacterium Ti plasmid or is derived from an Agrobacterium Ti plasmid. In yet another aspect, the vector provided herein is a viral vector.

[0116] On the one hand, plasmid or vector provided herein are recombinant vectors. As used herein, term " recombinant vector " refers to the vector formed by laboratory methods (such as molecular cloning) of genetic recombination. On the other hand, plasmid provided herein is a synthetic plasmid. As used herein, " synthetic plasmid " is an artificially produced plasmid that can have the same function (such as replication) as a natural plasmid (such as Ti plasmid). Without limitation, those skilled in the art can synthesize plasmids by single nucleotides, or by splicing together nucleic acid molecules from different pre-existing plasmids to produce synthetic plasmids from the beginning.

[0117] The carrier is commercially available or can be prepared by recombinant DNA techniques conventional in the art. On the one hand, provided herein is a carrier comprising all or part of SEQ ID NO:65. The carrier containing nucleic acid can have an expression element operably connected to this nucleic acid, and can also include a sequence (such as those (e.g., antibiotic resistance genes) encoding selectable markers). The carrier containing nucleic acid can encode a chimeric or fusion polypeptide (i.e., a polypeptide operably connected to a heterologous polypeptide, which can be located at the N-terminus or C-terminus of the polypeptide). Representative heterologous polypeptides are those (e.g., 6xHis tags, glutathione S-transferases (GST)) that can be used for purifying coded polypeptides.

[0118] Suitable methods for introducing polynucleotides (e.g., transgenes, recombinant vectors, recombinant DNA constructs, expression cassettes) into plant cells of the present disclosure include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Shillito et al. (1987) Meth. Enzymol. 153:313-336; Riggs et al. (1986) Proc. Natl. Acad. Sci. US 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763, 5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Org Culture Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926).See also Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (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); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, NY), pp. 197-209 (anther); Kaeppler et al. (1990) Plant Physiol. 27:175-182 (soybean); 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). On the one hand, the bacterial cells provided herein comprise the recombinant DNA constructs or recombinant vectors provided herein. It should be understood that many different types of bacterial cells can comprise recombinant DNA constructs or recombinant vectors, including but not limited to Agrobacterium tumefaciens and Escherichia coli. Yeast cells (e.g., Saccharomyces cerevisiae) comprising the recombinant DNA constructs or recombinant vectors provided herein are also provided.

[0119] On the other hand, recombinant construct or expression cassette provided herein can be introduced into plant by making plant contact with virus or viral nucleic acid.Usually, such method relates to expression cassette of the present disclosure being integrated into viral DNA or RNA molecule.It has been recognized that the promoter for expression cassette provided herein also encompasses promoters for transcription by viral RNA polymerase.Methods for introducing polynucleotide (including viral DNA or RNA molecule) into plant and expressing the protein encoded therein are known in the art.For example, referring to U.S. Patent number 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931 and Porta etc. (1996) Molecular Biotechnology 5:209-221.

[0120] Any plant tissue that can be propagated subsequently (no matter whether by organogenesis or embryogenesis) using recombinant constructs or expression cassettes provided herein can be transformed using cloning methods." Organogenesis" refers to the process of developing buds and roots from the center of the meristem in sequence. "Embryogenesis" refers to the process of buds and roots developing together from somatic cells or gametes in a coordinated manner (not sequentially). Exemplary tissues that are suitable for various transformation schemes as described herein include, but are not limited to, callus, existing meristems (such as shoot apical meristems, axillary buds and root meristems) and induced meristems (such as cotyledon meristems and hypocotyl meristems), hypocotyls, cotyledons, leaf discs, pollen, embryos, etc.

[0121] As generally understood in the art, the term "promoter" generally refers to a DNA sequence that comprises an RNA polymerase binding site, a transcription start site and / or a TATA box and assists or promotes the transcription and expression of a related transcribable polynucleotide sequence and / or a gene (or transgenic). Promoters can be synthesized, varied or derived from known or naturally occurring promoter sequences or other promoter sequences (e.g., as provided herein). Promoters can also include chimeric promoters comprising a combination of two or more heterologous sequences. Therefore, promoters of the present invention can include variants of promoter sequences that are similar in composition but not identical or complementary to other promoter sequences known or provided herein. As used herein, in the context of a DNA construct, a "heterologous promoter" refers to: (i) a promoter derived from a source different from an operably linked structural gene or coding region, or (ii) a promoter derived from a source identical to an operably linked structural gene or coding region, wherein the sequence of the promoter is modified from its original form. As used herein, the term "operably linked" refers to a functional connection between a promoter or other regulatory element and an associated transcribable polynucleotide sequence or a coding sequence of a gene (or transgene), such that the promoter, etc., initiates, assists, influences, causes, and / or promotes the transcription and expression of the associated coding or transcribable polynucleotide sequence, at least in specific tissues, developmental stages, and / or under certain conditions. A "plant-expressible promoter" refers to a promoter that can be used to express an associated coding sequence, transgene, or transcribable polynucleotide sequence operably linked to the promoter in plants, plant cells, and / or plant tissues.

[0122] Promoters can be classified according to various criteria related to the expression pattern of the sequence or gene (including transgenic) encoding the promoter operably linked thereto, such as constitutive, developmental, tissue-specific, inducible, etc. Promoters that initiate transcription in all or most tissues of a plant are referred to as "constitutive" promoters. Promoters that initiate transcription at certain periods or stages of development are referred to as "developmental" promoters. Promoters that increase expression in certain tissues of a plant relative to other plant tissues are referred to as "tissue-enhanced" or "tissue-preferred" promoters. Thus, "tissue-preferred" promoters cause relatively high or preferential expression in specific tissues of a plant, but lower expression levels in other tissues of the plant. Promoters that express in specific tissues of a plant but rarely express or do not express in other plant tissues are referred to as "tissue-specific" promoters. Promoters that express in a certain cell type of a plant are referred to as "cell-type-specific" promoters. "Inducible" promoters are promoters that initiate transcription in response to environmental stimuli, such as cold, drought, or light, or other stimuli (such as wounds or chemical applications). Promoters can also be classified according to their source, such as heterologous, homologous, chimeric, synthetic, etc. A "heterologous" promoter is a promoter sequence that has a different origin than its associated transcribable sequence, coding sequence, or gene (or transgene) and / or is not naturally present in the plant species to be transformed. When such a combination does not typically exist in nature, the term "heterologous" can refer more broadly to a combination of two or more DNA molecules or sequences. For example, if two or more DNA molecules or sequences are typically found in different genomes or at different loci in the same genome, or if they are not identically bound in nature, they will be heterologous to each other.

[0123] Exemplary constitutive promoters include the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); ubiquitin (Christensen et al. 1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J 3:2723-2730); the ALS promoter (U.S. Patent No. 5,659,026), etc.

[0124] Exemplary chemically inducible promoters include the tobacco PR-1a promoter activated by salicylic acid. Other chemically inducible promoters of interest include steroid-responsive promoters (e.g., see Schena et al. (1991) Proc. Natl. Acad. Sci. US 88: 10421-10425 and McNellis et al. (1998) Plant J. 14 (2): 247-257) glucocorticoid-inducible promoters) and tetracycline-inducible promoters (e.g., see Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237 and U.S. Patent numbers 5,814,618 and 5,789,156). Other exemplary promoters useful herein are those responsible for heat-regulated gene expression, light-regulated gene expression (e.g., pea rbcS-3A; maize rbcS promoter; the chlorophyll alb binding protein gene found in pea; or the Arabidopsis promoter), hormone-regulated gene expression (e.g., the abscisic acid (ABA) response sequence from the wheat Em gene; the ABA-inducible HVA1 and HVA22, and the rd29A promoters of barley and Arabidopsis; and wound-induced gene expression (e.g., wunl), organ-specific gene expression (e.g., tuber-specific storage protein genes; the described 23 kDa zein gene from maize; or French bean (β-phaseolin gene)), or pathogen-inducible promoters (e.g., the PR-1, prp-1, or β-1,3 glucanase promoters, the fungus-inducible wirla promoter of wheat, and the nematode-inducible promoters, TobRB7-5A and Hmg-1 of tobacco and parsley, respectively).

[0125] As used herein, a "leaf" promoter includes any promoter that initiates, causes, drives, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence in leaf tissue derived from any part of a plant. The "leaf" promoter can be further defined as one that initiates, causes, drives, etc., transcription or expression of its associated gene / transgene or transcribable DNA sequence in one or more tissues of a plant (e.g., one or more floral tissues). The "leaf" promoter can be further defined as a "leaf-preferred" promoter that at least preferentially or mostly (if not exclusively) initiates, causes, drives, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence in leaf tissue derived from any part of a plant (as opposed to floral tissue). However, "leaf" and "leaf-preferred" promoters can also each permit, allow, cause, drive, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence during the reproductive or developmental stages of one or more cells or tissues of a plant, such as one or more vegetative or reproductive tissues. Indeed, a "leaf" promoter may even initiate, cause, drive, etc., transcription or expression of its associated gene, transgene or transcribable DNA sequence in one or more reproductive or vegetative tissues at a greater level or extent than in leaf tissue.

[0126] As used herein, a "root" promoter includes any promoter that initiates, causes, drives, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence in root tissue derived from any part of a plant. The "root" promoter can be further defined as one that initiates, causes, drives, etc., transcription or expression of its associated gene / transgene, or transcribable DNA sequence in one or more tissues (e.g., one or more floral tissues) of a plant. The "root" promoter can be further defined as a "root-preferred" promoter that at least preferentially or mostly (if not exclusively) initiates, causes, drives, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence in root tissue derived from any part of a plant (as opposed to floral tissue). However, "root" and "root-preferred" promoters can also each permit, allow, cause, drive, etc., transcription or expression of its associated gene, transgene, or transcribable DNA sequence during the reproductive or developmental stages of one or more cells or tissues of a plant, such as one or more vegetative or reproductive tissues. Indeed, a "root" promoter may even initiate, cause, drive, etc., transcription or expression of its associated gene, transgene or transcribable DNA sequence in one or more reproductive or vegetative tissues at a greater level or extent than in root tissue.

[0127] Other exemplary tissue-preferred promoters include those disclosed in: 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. 112(3):1331-1341; VanCamp 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): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. US 90(20): 9586-9590; and Guevara-Garci et al. (1993) Plant J. 4(3): 495-505.

[0128] As used herein, "operably linked" refers to a functional connection between two or more elements. For example, an operable connection between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional connection that allows expression of the polynucleotide of interest. Operably linked elements can be continuous or discontinuous.

[0129] As used herein, "heterologous" refers to a sequence that is derived from an alien species, or, if derived from the same species, is substantially altered from its native form in the composition and / or genomic locus by intentional human intervention. The term also applies to nucleic acid constructs, also referred to herein as "polynucleotide constructs" or "nucleotide constructs." In this manner, a "heterologous" nucleic acid construct is intended to indicate that the construct is derived from an alien species, or, if derived from the same species, is substantially altered from its native form in the composition and / or genomic locus by intentional human intervention. For example, a heterologous nucleic acid construct includes, but is not limited to, a recombinant nucleotide construct that has been introduced into a plant or a plant part thereof by a transformation method or subsequent breeding of a transgenic plant with another target plant.

[0130] On the one hand, the expression of one or more polypeptides provided herein can be inhibited by RNA interference (RNAi) by expressing a polynucleotide provided herein. On the one hand, RNAi includes expressing non-coding RNA. As used herein, "non-coding RNA" is selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), trans-acting siRNA (ta-siRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), introns, hairpin RNA (hpRNA), intron-containing hairpin RNA (ihpRNA), and guide RNA. On the one hand, a single non-coding RNA provided herein inhibits the expression of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more than 10 polypeptides. On the one hand, the non-coding RNA provided herein is stably transformed into the plant genome. On the other hand, the non-coding RNA provided herein is transiently transformed into the plant genome.

[0131] As used herein, the terms "downregulate" and "inhibit" are defined as any method of reducing the expression or function of a target gene product (e.g., mRNA, protein, non-coding RNA) known in the art or as described herein. "Inhibit" can be compared between two plants (e.g., modified plants vs. control plants). Alternatively, the expression or function of the target gene product can be compared between plant cells, organelles, organs, tissues, or plant components within the same plant or between different plants, and include comparisons between developmental stages or time stages within the same plant or plant component or between plants or plant components. "Inhibit" includes any relative reduction in the function or production of the target gene product, up to and including complete elimination of the function or production of the gene product. The term "inhibit" encompasses any method or composition that downregulates the translation and / or transcription of the target gene product or the functional activity of the target gene product.

[0132] The term "inhibitory sequence" encompasses any polynucleotide or polypeptide sequence capable of inhibiting gene expression or function in a plant, such as a full-length polynucleotide or polypeptide sequence, a truncated polynucleotide or polypeptide sequence, a fragment of a polynucleotide or polypeptide sequence, a variant of a polynucleotide or polypeptide sequence, a nucleotide sequence in the sense orientation, a nucleotide sequence in the antisense orientation, a complementary sequence of a nucleotide sequence in the sense or antisense orientation, an inverted region of a nucleotide sequence, a hairpin of a nucleotide sequence, a double-stranded nucleotide sequence, a single-stranded nucleotide sequence, combinations thereof, etc. The term "polynucleotide sequence" includes sequences of RNA, DNA, chemically modified nucleic acids, nucleic acid analogs, combinations thereof, etc.

[0133] When the phrase "capable of inhibiting" is used in the context of a polynucleotide inhibitory sequence, it is intended that the inhibitory sequence itself exerts an inhibitory effect; or, when the inhibitory sequence encodes an inhibitory nucleotide molecule (e.g., a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or encodes an inhibitory polypeptide (e.g., a polypeptide that inhibits the expression or function of a target gene product), after its transcription (e.g., in the case of an inhibitory sequence encoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) or after its transcription and translation (in the case of an inhibitory sequence encoding an inhibitory polypeptide), the transcription or translation product, respectively, exerts an inhibitory effect on the target gene product (e.g., inhibits the expression or function of the target gene product).

[0134] The inhibitory sequences provided herein can be sequences that trigger gene silencing by any silencing pathway or mechanism known in the art, including but not limited to sense inhibition / co-suppression, antisense inhibition, double-stranded RNA (dsRNA) interference, hairpin RNA interference and intron-containing hairpin RNA interference, amplicon-mediated interference, ribozymes, small interfering RNA, artificial or synthetic microRNA, and artificial trans-acting siRNA. Depending on the desired outcome, the inhibitory sequence can be at least 20 nucleotides, at least 50 nucleotides, at least 70 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, up to the full-length polynucleotide encoding a protein of the present disclosure. In one aspect, the inhibitory sequence can be a fragment of 50-400 nucleotides, 70-350 nucleotides, 90-325 nucleotides, 90-300 nucleotides, 90-275 nucleotides, 100-400 nucleotides, 100-350 nucleotides, 100-325 nucleotides, 100-300 nucleotides, 125-300 nucleotides, or 125-275 nucleotides in length.

[0135] MicroRNA (miRNA) is a non-protein coding RNA, usually between 19-25 nucleotides (usually 20-24 nucleotides in plants), which guides the trans-cleavage of target transcripts, negatively regulating the expression of genes involved in various regulatory and developmental pathways (Bartel (2004) Cell, 116: 281-297). In some cases, miRNA is used to guide the synchronous processing of siRNA primary transcripts (see Allen et al. (2005) Cell, 121: 207-221).

[0136] Many microRNA genes (MIR genes) have been identified and are publicly available in databases ("miRBase", available online at microrna.sanger.ac.uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes are reported to occur in intergenic regions (isolated and clustered in the genome), but may also be located entirely or partially in introns of other genes (protein-coding and non-protein-coding). At least in some cases, transcription of MIR genes can be under the facilitating control of the MIR gene's own promoter. The primary transcript, called "pri-miRNA", can be quite large (several thousand bases) and can be polycistronic, containing one or more pre-miRNAs (a folded structure containing a stem-loop arrangement that is processed into mature miRNAs) and the 5' "cap" and polyadenylation tail common to mRNAs.

[0137] The maturity of mature miRNAs from their corresponding precursors (pri-miRNA and pre-miRNA) varies significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are thought to be fully processed into mature miRNAs primarily in the nucleus, while in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, and then pre-miRNAs are exported to the cytoplasm and further processed into mature miRNAs. The length of mature miRNAs in plants is typically 21 nucleotides.

[0138] Transgenic expression of miRNA (whether naturally occurring sequence or artificial sequence) can be used to regulate the expression of one or more target genes of miRNA. Including miRNA recognition sites in transgenic expressed transcripts can also be used to regulate the expression of transcripts; For example, see Parizotto et al. (2004) Genes Dev., 18: 2237-2242. The recognition sites of miRNA have been verified in all regions of mRNA, including 5' untranslated regions, coding regions and 3' untranslated regions, indicating that the position of miRNA target sites relative to the coding sequence may not necessarily affect inhibition. Because miRNA is an important regulatory element in eukaryotes, transgenic inhibition of miRNA can be used to manipulate biological pathways and reactions. Finally, the promoter of the MIR gene can have a very specific expression pattern (such as cell-specific, tissue-specific, time-specific or inducible), so it can be used for recombinant constructs to induce this specific transcription of the DNA sequence operably connected to them. The various uses of miRNA, their precursors, their recognition sites and their promoters are known. Non-limiting examples of these uses include: (1) expressing a natural miRNA or miRNA precursor sequence to inhibit a target gene; (2) expressing an artificial miRNA or miRNA precursor sequence to inhibit a target gene; (3) expressing a transgene with a miRNA recognition site, wherein the transgene is inhibited when the mature miRNA is expressed; and (4) expressing a transgene driven by a miRNA promoter.

[0139] Designing artificial miRNA sequences can be as simple as replacing nucleotides in the miRNA stem region of the miRNA precursor with sequences complementary to the intended target. A non-limiting example of a general method for determining nucleotide changes in a natural miRNA sequence to prepare an engineered miRNA precursor includes the following steps: (a) selecting a unique target sequence of at least 18 nucleotides that is specific for the target gene, for example, by using a sequence alignment tool such as BLAST of tobacco cDNA and genomic DNA databases (see, e.g., Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402) to identify target transcript orthologs and any potential matches to unrelated genes, thereby avoiding inadvertent silencing of non-target sequences; (b) analyzing the target gene for unwanted sequences (e.g., matches to sequences from non-target species) and scoring each potential 19-mer fragment for the following: GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22:326-330) and functional asymmetry characterized by a negative difference in free energy (".DELTA..DELTA.G" or "ΔΔG"). 19-mers having all or most of the following characteristics are preferably selected: (1) Reynolds score > 4, (2) GC content of 40%-60%, (3) negative ΔΔG, (4) terminal adenosine, (5) lack of 4 or more consecutive identical nucleotides; (6) located near the 3' end of the target gene; and (7) minimal difference from the miRNA precursor transcript. It has been reported that the position of each third nucleotide in the siRNA is particularly important in affecting RNAi efficacy. The algorithm "siExplorer" is publicly available at rna.chem.tu-tokyo.ac.jp / siexplorer.htm; (c) determining the reverse complementary sequence of the selected 19-mer for use in preparing modified mature miRNAs. The other nucleotide at position 20 is preferably matched to the selected target sequence, and the nucleotide at position 21 is preferably selected not to be paired to prevent the spread of silencing on the target transcript, or is selected to pair with the target sequence to promote the spread of silencing on the target sequence; and (d) introducing the artificial miRNA into the plant.

[0140] In one aspect, the artificial miRNA provided herein reduces or eliminates RNA transcription or protein translation of a target gene.

[0141] In one aspect, the miRNA or artificial miRNA provided herein is under the control of a tissue-specific promoter. In a further aspect, the miRNA or artificial miRNA provided herein is under the control of a tissue-preferred promoter. In a further aspect, the miRNA or artificial miRNA provided herein is under the control of a constitutive promoter.

[0142] Tobacco materials obtained from the modified tobacco strains, varieties, or hybrids disclosed herein can be used to prepare tobacco products. As used herein, "tobacco product" is defined as any product prepared or derived from tobacco intended for human use or consumption. In one aspect, the tobacco products provided herein comprise cured components from the modified tobacco plants provided herein. In another aspect, the tobacco products provided herein comprise cured tobacco leaves from the modified tobacco plants provided herein.

[0143] Tobacco products provided herein include, but are not limited to, tobacco products (e.g., cigarettes, bidis, kreteks), cigar products (e.g., cigars, cigar wrappers, cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, chewing tobacco, moist smokeless tobacco, fine-cut chewing tobacco, long-cut chewing tobacco, pouched chewing tobacco), films, chewables (e.g., chewing gum), lozenges, dissolving strips, labels, tablets, shaped portions, gels, consumer product units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, for example, U.S. Patent Publication No. US 2006 / 0191548.

[0144] As used herein, "cigarette" refers to a tobacco product having a "rod" and a "filler." The cigarette "rod" includes the cigarette paper, the filter, the filter plug wrap (for containing the filter material), the tipping paper that secures the cigarette paper (including the filler) to the filter, and any glue that holds these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other flavorings that may accompany tobacco in the cigarette paper), (3) the casing, (4) flavorings, and (5) all other additives (mixed with tobacco and substitutes and rolled into the cigarette).

[0145] In one aspect, the present disclosure provides derivatized nicotine and methods of producing nicotine for use in articles of manufacture from the modified tobacco plants provided herein.

[0146] The tobacco product derived from plant of the present disclosure also comprises cigarettes and other smoking products, particularly those smoking products that comprise filter elements, wherein the smokable material rod comprises the cured tobacco in the tobacco blend.On the one hand, tobacco product of the present disclosure is selected from lower group: cigarillos, non-ventilated filter cigarettes, ventilated filter cigarettes, bidis, cigars, snuff, pipe tobacco, cigar leaves, cigarette leaves, chewing tobacco, whole tobacco leaves, hookah, shredded tobacco (shredded tobacco) and raw cut tobacco (cut tobacco).On the other hand, tobacco product of the present disclosure is a smokeless tobacco product.Smokeless tobacco product does not burn, and includes but is not limited to chewing tobacco, moist smokeless tobacco, snuff and dry snuff.Chewing tobacco is the tobacco leaf of coarse division, and it is typically packaged in large bag-shaped packages and is used for strips (plug) or slices (twist). Moist smokeless tobacco is a moist, more finely divided tobacco that is provided in a loose form or in the form of a pouch and is typically packaged in a round can and used as a pinch or packaged in a pouch placed between the cheek and gums of an adult tobacco consumer. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco that is placed in the mouth or used for the nasal cavity. In a further aspect, the tobacco product of the present disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of electronically heated cigarettes, e-cigarettes, and electronic vaporization devices. On the one hand, the method provided herein comprises preparing a tobacco product using cured tobacco leaves from a modified tobacco plant provided herein.

[0147] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco powder and other tobacco waste that is processed into sheets and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is important because it contributes to the taste of cigarettes by processing flavor development using a reaction between ammonia and sugars.

[0148] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been treated by expansion with a suitable gas, causing the tobacco to "expand," resulting in a decrease in density and an increase in filling power. It reduces the weight of the tobacco used in cigarettes.

[0149] This paper also provides a cured tobacco material made from tobacco plants or plant components provided herein. "Curing" is a aging process that reduces moisture and causes the destruction of chlorophyll, making tobacco leaves appear golden yellow, and starch is also converted into sugar. Therefore, compared with the green leaves of the harvest, cured tobacco has a higher reducing sugar content and a lower starch content. On the one hand, conventional methods can be used to cure tobacco plants or plant components provided herein, such as curing, barn curing, open fire curing, air-curing or sun-curing. For the description of various types of curing methods, for example, see Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is usually aged for several years (e.g., 2-5 years) in wooden barrels (e.g., vats) or cardboard boxes under compression conditions with a moisture content of 10%-25%. Referring to U.S. Patent Nos. 4,516,590 and 5,372,149. Then, the cured and aged tobacco can be further processed. Further processing includes modulating tobacco under vacuum with or without the introduction of steam, pasteurization and fermentation at various temperatures. Typically, fermentation is characterized by a high initial moisture content, heat generation and a dry weight loss of 10-20%. For example, referring to U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, aged and fermented tobacco can be further processed (e.g., shredded, chopped, puffed or mixed). See, for example, US Patent Nos. 4,528,993; 4,660,577; and 4,987,907. In one aspect, the cured tobacco material of the present disclosure is flue-cured, sun-cured, air-cured, or flame-cured.

[0150] The disclosure also provides a method for manufacturing tobacco products, and the tobacco products comprise tobacco materials from tobacco plants provided herein. On the one hand, the method provided herein comprises modulating the aged tobacco materials made from tobacco plants provided herein, so that its moisture content is increased from 12.5%-13.5% to 21%, mixing the modulated tobacco materials to produce required mixture. On the one hand, the method for preparing tobacco products provided herein further comprises carrying out feeding (casing) or seasoning to the mixture. Usually, in the feeding process, the material of feeding or seasoning is added in the mixture, so as to improve their quality and develop some required flavor characteristics by balancing chemical composition. The further details of the feeding process can be found in Tobacco Production, Chemistry and Technology, Blackwell Science, 1999, edited by L. Davis and M. Nielsen.

[0151] Also can use but not limited to heat treatment (for example cooking, baking), seasoning, enzyme treatment, puffing and / or curing method processing tobacco material provided herein.Can use these technical processing fermentation and non-fermented tobacco.The example of suitable processing tobacco comprises dark air-cured, dark fire-cured, burley, cured and cigar filler or wrapper, and the product of the operation of the stem from all leaves.On the one hand, tobacco fiber comprises based on fresh weight up to 70% dark tobacco.For example, as described in U.S. Publication No. 2004 / 0118422 or 2005 / 0178398, can be by heating, fermentation (sweating) and / or pasteurization step to modulate tobacco.

[0152] Tobacco material provided herein can be fermented. Typically, the feature of fermentation is that initial moisture content is high, produces heat and dry weight loss 10-20%. For example, referring to U.S. Patent number 4,528,993; 4,660,577; 4,848,373 and 5,372,149. Except changing the fragrance of leaf, fermentation can also change the color and texture of leaf. Equally in fermentation process, evolution gas can be produced, oxygen can be absorbed, pH value can be changed, the amount of water retained can be changed. For example, referring to U.S. Publication No. 2005 / 0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Before being incorporated into oral products, the tobacco of (for example shredding, puffing, mixing, grinding or pulverizing) curing or curing and fermentation can be further processed. In some cases, the tobacco is long cut fermented cured wet tobacco having an oven volatiles content of 48-50% by weight prior to mixing with the copolymer and optional flavorants and other additives.

[0153] On the one hand, tobacco material provided herein can be processed into required size.In some aspects, tobacco fiber can be processed into average fiber size less than 200 microns.On the one hand, tobacco fiber is the 75-125 micron.On the other hand, tobacco fiber is processed into 75 microns or littler of a size.On the one hand, tobacco fiber comprises long cut tobacco, and it is 10 cuts / inch (cut / inch) until 110 cuts / inch that it can be shredded or chopped into width, and length is 0.1 inch until 1 inch.Double cut tobacco fiber can have the particle size of certain limit, makes 70% double cut tobacco fiber fall between the mesh size of-20 order-80 order.

[0154] The tobacco material provided herein can be processed to have a total oven volatiles content of 10 wt % or more; 20 wt % or more; 40 wt % or more; 15 wt %-25 wt %; 20 wt %-30 wt %; 30 wt %-50 wt %; 45 wt %-65 wt % or 50 wt %-60 wt %. It will be understood by those skilled in the art that "moist" tobacco typically refers to tobacco having an oven volatiles content of about 40 wt %-60 wt % (e.g., 45 wt %-55 wt %, or 50% wt). As used herein, "oven volatiles" are determined by calculating the percent weight loss of a sample after drying it at 110° C. for 3.25 hours in a preheated forced air oven. The total oven volatiles content of an oral product may be different from the oven volatiles content of the tobacco fiber used to prepare the oral product. The processing steps described herein can reduce or increase the oven volatiles content.

[0155] In one aspect, the tobacco plants, seeds, plant components, plant cells, and plant genomes provided herein are from a tobacco type selected from the group consisting of: flue-cured tobacco, sun-cured tobacco, air-cured tobacco, dark air-cured tobacco, and dark fire-cured tobacco. In another aspect, the tobacco plants, seeds, plant components, plant cells, and plant genomes provided herein are from a tobacco type selected from the group consisting of: burley, Maryland, golden, Virginia, Oriental, Turkish, and Tobacco. On the one hand, the tobacco plant or seed provided herein is a hybrid plant or seed. As used herein, "hybrids" are produced by hybridizing two plants from different varieties or species so that offspring comprise the genetic material from each parent. Skilled artisans recognize that higher order hybrids can also be generated. For example, a first hybrid can be prepared by hybridizing variety C with variety D to produce a C x D hybrid, and a second hybrid can be produced by hybridizing variety E with variety F to produce an E x F hybrid. The first and second hybrids can be further hybridized to produce a higher order hybrid (C x D) x (E x F) comprising the genetic information from all four parental varieties.

[0156] Flue-cured tobacco (also known as Virginia or golden tobacco) accounts for approximately 40% of the world's tobacco production. Because it develops a golden to deep orange color during curing, flue-cured tobacco is often referred to as "golden tobacco." Flue-cured tobacco has a light, bright aroma and flavor. Flue-cured tobacco is typically high in sugar and low in oil. Major flue-cured tobacco-growing countries include Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, the modified tobacco plant or seed provided herein is a flue-cured tobacco background selected from the group consisting of CC 13, CC 27, CC 33, CC35, CC 37, CC 65, CC67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety substantially derived from any one of the above varieties. In another aspect, the modified tobacco plants or seeds provided herein are of a flue-cured tobacco background selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF, NC 55, NC60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair 373, McNair944, Ox 207, Ox 414NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG Speight NF-3, V116, V182, and any variety substantially derived from any of the foregoing. See WO 2004 / 041006 A1.In a further aspect, the modified tobacco plants, seeds, hybrids, varieties or lines provided herein are of any flue-cured background selected from the group consisting of K326, K346 and NC196.

[0157] Air-cured tobaccos include Burley, Maryland, and Dark. They all share a common characteristic: they are cured primarily without artificial heat and humidity. Burley tobacco is light to dark brown in color, high in oil and low in sugar. Burley tobacco is air-cured in barns. Major Burley tobacco-growing countries include Argentina, Brazil, Italy, Malawi, and the United States. Maryland tobacco is extremely fluffy, has good burning properties, is low in nicotine, and has a neutral aroma. Major Maryland tobacco-growing countries include the United States and Italy. In another aspect, the modified tobacco plant or seed provided herein is of a Burley background selected from the group consisting of Clay 402, Clay403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, HB4488PLC, PD 7319LC, Bu 21×Ky 10, HB04P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561, and V509. In a further aspect, the modified tobacco plants, seeds, hybrids, varieties or lines provided herein are any Burley background selected from the group consisting of TN 90, KT209, KT 206, KT212 and HB 4488. In another aspect, the modified tobacco plants or seeds provided herein are any Maryland background selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341.

[0158] Dark air-cured tobacco differs primarily from other types in the curing process, which imparts its brown to dark brown color and distinct aroma. Dark air-cured tobacco is primarily used in the production of chewing tobacco and snuff. In one aspect, the modified tobacco plants or seeds provided herein are dark air-cured tobaccos selected from the group consisting of Sumatra, Jatim, Dominic Cubano, Besuki, One Sucker, Green River, Virginis un-cured, and Paragu Passado.

[0159] Dark flame-cured tobacco is typically cured over a low burning wood fire on the floor of a closed curing room. Dark flame-cured tobacco is used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong cigars. The main growing regions for dark flame-cured tobacco are Tennessee, Kentucky, and Virginia in the U.S. On the one hand, the modified tobacco plant or seed provided herein is a dark flame-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF485, TN D94, TN D950, V309, and V359.

[0160] Oriental tobacco is also referred to as Greek aroma and Turkish tobacco, because they are typically planted in the eastern Mediterranean region, for example Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy and Romania.The feature of today's Oriental kind is that the size of plant and leaf is little, and its unique aroma characteristic, and this is the result that plant adapted to poor soil and adverse climatic conditions in many centuries in the past.On the one hand, modified tobacco plant provided herein or seed are the Oriental tobacco background that are selected from lower group: Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and basically derived from any one of any kind of above-mentioned kind.

[0161] In one aspect, the modified tobacco plants, seeds, hybrids, varieties, or lines provided herein are substantially derived from or have a genetic background selected from the group consisting of: BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, GL 26H, GL 338, GL350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326, NC95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, V116, V119, KDH 959, KT200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY 907LC, KTY14 x L8 LC, Little Crittenden, McNair 373, McNair 944, male sterile KY 14x L8, Narrow Leaf Madole, MS KY171, Narrow Leaf Madole(phph), MS Narrow LeafMadole, MS TND950, PD 7302LC, PD7305LC, PD 7309LC, PD 7312LC, PD 7318LC, PD 7319LC, MSTKS 2002, TKF 2002, TKF 6400, TKF 4028, TKF 4024, KT206LC, KT209LC, KT210LC, KT212LC, NC 100, NC 102, NC 2000, NC291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TND950, TR (Tom Rosson) Madole, V309, V359, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art.

[0162] All of the aforementioned specific varieties of dark air-cured, Burley, Maryland, dark fire-cured, or Oriental types are listed for exemplary purposes only. Any other dark air-cured, Burley, Maryland, dark fire-cured, or Oriental varieties are also contemplated in this application.

[0163] This paper also provides the colony of tobacco plant as herein described.On the one hand, the planting density of tobacco plant colony provided herein is 5,000-8000,5,000-7,600,5,000-7,200,5,000-6,800,5,000-6,400,5,000-6,000,5,000-5,600,5,000-5,200,5,200-8,000,5,600-8,000,6,000-8,000,6,400-8,000,6,800-8,000,7,200-8,000 or 7,600-8,000 strain plant.

[0164] This paper also provides a container for the seed from tobacco plant described herein. The container of tobacco seeds of the present disclosure can include seeds of any quantity, weight or volume. For example, the container can include at least or more than 10 seeds; at least or more than 25 seeds; at least or more than 50 seeds; at least or more than 100 seeds; at least or more than 200 seeds; at least or more than 300 seeds; at least or more than 400 seeds; at least or more than 500 seeds; at least or more than 600 seeds; at least or more than 700 seeds; at least or more than 800 seeds; at least or more than 900 seeds; at least or more than 1000 seeds; at least or more than 1500 seeds; at least or more than 2000 seeds; at least or more than 2500 seeds; at least or more than 3000 seeds; at least or more than 3500 seeds; at least or more than 4000 seeds; or at least or more than 5000 seeds. Alternatively, the container can contain at least or more than 1 ounce of seeds; at least or more than 5 grams of seeds; at least or more than 10 grams of seeds; at least or more than 30 grams of seeds; at least or more than 50 grams of seeds; at least or more than 100 grams of seeds; at least or more than 500 grams of seeds; at least or more than 1 kilogram of seeds; at least or more than 1.5 kilograms of seeds; at least or more than 2 kilograms of seeds; at least or more than 5 kilograms of seeds; or at least or more than 10 kilograms of seeds. The container of tobacco seeds can be any container available in this area. As non-limiting examples, the container can be a box, bag, packet, pouch, roll, tube or bottle.

[0165] In one aspect, the present disclosure provides cured leaves from a modified tobacco plant comprising reduced levels of one or more TSNAs. In one aspect, the reduced one or more TSNAs are selected from the group consisting of N'-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK) / N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and any combination thereof. In one aspect, the levels of total or individual TSNAs are measured on freeze-dried, cured leaf samples using liquid chromatography with tandem mass spectrometry (LC / MS / MS).

[0166] In one aspect, the present disclosure provides cured leaves from a modified tobacco plant comprising reduced levels of one or more alkaloids. In one aspect, the reduced one or more alkaloids are selected from the group consisting of nicotine, nornicotine, anabasine, and anatabine.

[0167] The disclosure also provides the method for the tobacco strain, cultivar or kind that comprises the nitrogen utilization efficiency of enhancement for breeding.Can be carried out breeding by any known program.DNA fingerprint analysis, SNP mapping, haplotype mapping or similar technology can be used for marker assisted selection (MAS) breeding program, with desired proterties or allele transfer or breeding in tobacco plant.For example, the breeder can use the F1 hybrid plant provided herein to produce segregating population in F2 or backcross generation, or further make the F1 hybrid plant hybridize with other donor plants with the genotype desired on agronomy.Can use one of technology known in the art or listed herein to screen the agronomy proterties or the expected chemical spectrum of the expectation of F2 or backcross generation plant.Depend on the genetic pattern of expectation or the MAS technology used, can before each backcross cycle, selected plant is carried out self-pollination to help identify required individual plant.Can repeat backcross or other breeding programs, until recover the desired phenotype of recurrent parent. In one aspect, the recurrent parent of the present disclosure can be a cured variety, a white rib variety, a dark air-cured variety, a dark flame-cured variety, or an oriental variety. In another aspect, the recurrent parent can be a modified tobacco plant, line, or variety. In one aspect, the recurrent parent provided herein is TN90. In another aspect, the recurrent parent provided herein is MD609. For example, other breeding techniques can be found in Wernsman, EA, and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. WH Fehr (ed.), MacMill Publishing Co., Inc., New York, NY, which is incorporated herein by reference in its entirety.

[0168] The result of the plant breeding program using modified tobacco plant as herein described comprises useful strain, cultivar, kind, offspring, inbred line and hybrid of the present disclosure.As used herein, term " kind " refers to the plant colony with the constant characteristic that separates it from other plants of the same species.Kind is normally (although not always) commercial sale.When having one or more special traits, the further feature of kind is very little overall difference between individual in this kind.Can be by several generations of self-pollination and selection, or use tissue or cell culture technique to generate " pure line " kind from single parent asexual propagation.Kind can be derived from another strain or kind basically. According to the definition of the International Convention for the Protection of New Varieties of Plants (December 2, 1961, revised in Geneva on November 10, 1972, October 23, 1978 and March 19, 1991), variety is "basically derived" from initial variety, if: a) it is mainly derived from initial variety, or derived from a variety mainly derived from initial variety, while retaining the expression of the essential characteristics produced by the genotype of the initial variety or the genotype combination; b) it is significantly different from the initial variety; c) except the difference produced by the derivation behavior, it conforms to the initial variety in the expression of the essential characteristics produced by the genotype of the initial variety or the genotype combination. For example, the variety derived basically can be obtained by selecting a natural or induced mutant, somatic cell asexual variant, a variation individual, backcrossing or conversion from the initial variety plant. It is believed that the first tobacco variety and the first variety have substantially the same genetic background basically from the second tobacco variety derived therefrom. Different from variety, " strain " most typically refers to a group of plants used for non-commercial purposes, for example, for plant research. Lines typically display very little overall variation among individuals in one or more traits of interest, although there may be some variation among individuals in other traits.

[0169] On the one hand, present disclosure provides a kind of method of producing tobacco plant, comprise at least one tobacco plant of the first tobacco variety and at least one tobacco plant of the second tobacco variety, wherein compared with the control tobacco plant of the same variety grown under comparable conditions, at least one tobacco plant of the first tobacco variety shows enhanced nitrogen use efficiency; Select and compare with the control tobacco plant of the same hybridization grown under comparable conditions, show the offspring tobacco plant of enhanced nitrogen use efficiency.On the one hand, the first tobacco variety provided herein comprises modified tobacco plant.On the other hand, the second tobacco variety provided herein comprises modified tobacco plant.On the one hand, the first or second tobacco variety is male sterile.On the other hand, the first or second tobacco variety is cytoplasmic male sterile.On the other hand, the first or second tobacco variety is female sterile.On the one hand, the first or second tobacco variety is an improved variety.On the other hand, the first or second tobacco variety is a hybrid.

[0170] On the one hand, present disclosure provides a kind of method that one or more transgenics are infiltrated into tobacco variety, the method comprises: (a) will comprise the first tobacco variety of one or more transgenics and do not contain the second tobacco variety of described one or more transgenics hybridize to produce one or more offspring tobacco plants; (b) one or more offspring tobacco plants are carried out to one or more transgenic genotyping; (c) select to comprise the offspring tobacco plants of one or more transgenics.On the other hand, these methods further comprise and make selected offspring tobacco plant and the second tobacco variety backcross.In further aspect, these methods also comprise: (d) will select the offspring plant of selection and itself or with the second tobacco variety hybridize to produce one or more further offspring tobacco plants; (e) select to comprise the further offspring tobacco plants of one or more transgenics.On the one hand, the second tobacco variety is improved varieties.

[0171] On the one hand, present disclosure provides a kind of method that one or more sudden changes are infiltrated into tobacco variety, the method comprises: (a) will comprise the first tobacco variety of one or more sudden changes and do not contain the second tobacco variety of described one or more sudden changes to produce one or more offspring tobacco plants; (b) one or more offspring tobacco plants are carried out the genotyping of one or more sudden changes; and (c) select the offspring tobacco plant that comprises one or more sudden changes.On the other hand, these methods also comprise and make selected offspring tobacco plant and the second tobacco variety backcross.In further aspect, these methods also comprise: (d) will select the offspring plant and itself or with the second tobacco variety hybrid to produce one or more further offspring tobacco plants; and (e) select to comprise the further offspring tobacco plant of one or more sudden changes.On the one hand, the second tobacco variety is improved varieties.

[0172] In one aspect, the present disclosure provides a method of growing a population of modified tobacco plants comprising enhanced nitrogen use efficiency, wherein the method comprises planting a population of tobacco seeds comprising one or more mutations, one or more transgenes, or both, wherein the one or more modified tobacco plants exhibit enhanced nitrogen use efficiency when compared to a control tobacco plant of the same variety when grown under comparable conditions.

[0173] In one aspect, the present disclosure provides a method for preparing modified seeds, comprising introducing a recombinant DNA construct provided herein into a plant cell; screening a population of plant cells for the recombinant DNA construct; selecting one or more plant cells from the population; generating one or more modified plants from the one or more plant cells; and collecting one or more modified seeds from the one or more modified plants.

[0174] As used herein, a "locus" is a chromosomal region where a polymorphic nucleic acid, a trait determinant, a gene or a marker is located. The locus of the present disclosure comprises one or more polymorphisms in a population; for example, there are alternative alleles in some individuals. As used herein, an "allele" refers to an alternative nucleic acid sequence at a specific locus. The length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, for example, for different chromosomes in a heterozygous individual, or between different homozygous or heterozygous individuals in a population. As used herein, a chromosome in a diploid plant is "hemizygous" when there is only one copy of the locus. For example, when an inserted transgene is inserted into only one sister chromosome (i.e., the second sister chromosome does not contain the inserted transgene), the inserted transgene is hemizygous.

[0175] In one aspect, the plant, seed, plant component, plant cell or plant genome of the modification is homozygous for the transgene provided herein. In another aspect, the plant, seed, plant component, plant cell or plant genome of the modification is heterozygous for the transgene provided herein. In one aspect, the plant, seed, plant component, plant cell or plant genome of the modification is hemizygous for the transgene provided herein. In one aspect, the plant, seed, plant component, plant cell or plant genome of the modification is homozygous for the mutation provided herein. In another aspect, the plant, seed, plant component, plant cell or plant genome of the modification is heterozygous for the mutation provided herein. In one aspect, the plant, seed, plant component, plant cell or plant genome of the modification is hemizygous for the mutation provided herein.

[0176] As used herein, "introgression" refers to the transmission of a desired allele of a genetic locus from one genetic background to another.

[0177] As used herein, "crossing" refers to the production of offspring (eg, cells, seeds, or plants) through fertilization, and includes crossing between different plants (sexual) and self-fertilization (selfing).

[0178] As used herein, " backcross " refers to the process that offspring plant and one of its parents repeat hybridization.In the backcross scheme, " donor " parent refers to the parent plant with required gene to be infiltrated or locus." recipient " parent (using once or repeatedly) or " recurrent " parent (using twice or more times) refer to the parent plant that gene or locus are infiltrated therein.Initial hybridization produces F1 generation.Term " BC1 " refers to use recurrent parent for the second time, " BC2 " refers to use recurrent parent for the third time, and the rest may be deduced by analogy.On the one hand, repeat backcross, the offspring individual self of each continuous backcross generation is backcrossed with identical parental genotype.

[0179] As used herein, "elite variety" refers to any variety resulting from breeding and selection due to superior agronomic performance.

[0180] As used herein, "selection" in the context of breeding refers to the act of culling or choosing desired individuals, usually from a population, based on certain predetermined criteria.

[0181] On the one hand, tobacco plant provided herein is a hybrid plant. Hybrids can be produced by: stopping the female parent plant (for example seed parent) of the first kind from pollination, allowing the pollen fertilization female parent plant of the male parent plant of the second kind, and allowing F1 hybrid seeds to form on female plants. The self-pollination of female plants can be stopped by emasculating flowers in the early stages of flower development. Alternatively, male sterile forms can be used to stop pollen formation on female parent plants. For example, male sterility or self-incompatibility can be produced by male sterility (MS) or wherein transgenic suppression of microspore formation and / or pollen formation. The female parent plant comprising MS is particularly useful. Aspects where the female parent plant is MS, pollen can be gathered in the crops from male fertile plants, and artificially fertilized to the stigma of the MS female parent plant, and the F1 seed of the crops gained. In addition, female sterile plants can also be used to stop self-pollination.

[0182] Plant can be used for forming single cross tobacco F1 hybrid.To be artificially transferred to the female parent plant of emasculation or the female parent plant of male sterility to form F1 seed from the pollen of male parent plant.Perhaps, can carry out three-way hybridization, wherein single cross F1 hybrid is as female parent and hybridizes with different male parents.As another selection, can produce double cross hybrid, wherein the F1 offspring of two different single crosses hybridizes with themselves.When forming double cross hybrid, self-incompatibility can be used to prevent the self-pollination of female parent especially advantageously.

[0183] On the one hand, tobacco variety provided herein is male sterility. On the other hand, tobacco variety provided herein is cytoplasmic male sterility (CMS). Male sterile tobacco plants can be produced by any method known in the art. The method for producing male sterile tobacco is described in Wernsman, EA, and Rufty, RC1987.Chapter Seventeen.Tobacco.Pages 669-698In:Cultivar Development.Crop Species.WH Fehr (ed.), MacMill Publishing Go., Inc., New York, NY761pp. On the other hand, tobacco variety provided herein is female sterility. As a non-limiting example, female sterile plants can be prepared by sudden change STIG1 gene. For example, referring to Goldman et al. 1994, EMBO Journal 13:2976-2984.

[0184] In one aspect, the present disclosure provides and encompasses a method for determining the NUE of a tobacco line, comprising obtaining at least one metabolite from a tobacco plant of the tobacco line, determining the amount of the at least one metabolite obtained, and determining the NUE of the tobacco line based on the amount of the at least one metabolite determined. In a further aspect, the at least one metabolite is obtained from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue. In a further aspect of the method, at least two metabolites are obtained. In a further aspect of the method, at least three metabolites are obtained. In a further aspect of the method, at least four metabolites are obtained. In a further aspect of the method, at least five metabolites are obtained. In a further aspect of the method, at least six metabolites are obtained. In a further aspect of the method, at least seven metabolites are obtained. In a further aspect of the method, at least eight metabolites are obtained. In a further aspect of the method, at least nine metabolites are obtained. In a further aspect of the method, at least ten metabolites are obtained. In a further aspect of the method, the amounts of at least two metabolites are determined. In a further aspect of the method, the amounts of at least three metabolites are determined. In a further aspect of the method, the amounts of at least four metabolites are determined. In a further aspect of the method, the amounts of at least five metabolites are determined. In a further aspect of the method, the amounts of at least six metabolites are determined. In a further aspect of the method, the amounts of at least seven metabolites are determined. In a further aspect of the method, the amounts of at least eight metabolites are determined. In a further aspect of the method, the amounts of at least nine metabolites are determined. In a further aspect of the method, the amounts of at least ten metabolites are determined.

[0185] In another aspect of the methods provided herein, the amount of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, D-23937, X-23937, X-23916, 1-methyladenine, 4-guanidinobutanoate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, X-23366, N-acetylphenylalanine, naringenin, X-23454, X-23580, and X-23852 is determined.

[0186] In another aspect of the methods provided herein, a tobacco plant with enhanced NUE comprises enhanced NUE in at least one tissue, compared to a tobacco plant that comprises a lower amount of at least one metabolite. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least one metabolite in two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least four metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least five metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least one metabolite in three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least two metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least three metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least four metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least five metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least one metabolite in four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least two metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least three metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least four metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least five metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least one metabolite in five tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least two metabolites in at least five tissues.In a further aspect, the tobacco plant with enhanced NUE contains lower amounts of at least three metabolites in at least five tissues. In a further aspect, the tobacco plant with enhanced NUE contains lower amounts of at least four metabolites in at least five tissues. In a further aspect, the tobacco plant with enhanced NUE contains lower amounts of at least five metabolites in at least five tissues.

[0187] In another aspect of the methods provided herein, a tobacco plant with enhanced NUE comprises enhanced NUE in at least one tissue, compared to a tobacco line that comprises a higher amount of at least one metabolite. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least one metabolite in two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least four metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least five metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least one metabolite in three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least two metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least three metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least four metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least five metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least one metabolite in four tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least two metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least three metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least four metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least five metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least one metabolite in five tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least two metabolites in at least five tissues.In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least three metabolites in at least five tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least four metabolites in at least five tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least five metabolites in at least five tissues.

[0188] In another aspect of the methods provided herein, a tobacco plant with enhanced NUE comprises enhanced NUE in at least one tissue compared to a tobacco line that comprises an equivalent amount of at least one metabolite. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least one metabolite in three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least four metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least five metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least one metabolite in four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least two metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least three metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least four metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least five metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least one metabolite in five tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least two metabolites in at least five tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least three metabolites in at least five tissues.In a further aspect, the tobacco plant with enhanced NUE comprises equal amounts of at least four metabolites in at least five tissues. In a further aspect, the tobacco plant with enhanced NUE comprises equal amounts of at least five metabolites in at least five tissues.

[0189] In another aspect of the methods provided herein, a tobacco plant with enhanced NUE comprises reduced NUE in at least one tissue compared to a tobacco line that comprises a lower amount of at least one metabolite. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least one metabolite in two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises lower amounts of at least four metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least five metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least one metabolite in three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least two metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least three metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least four metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least five metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least one metabolite in four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least two metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least three metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least four metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least five metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least one metabolite in five tissues. In a further aspect, tobacco plants with enhanced NUE contain lower amounts of at least two metabolites in at least five tissues.In a further aspect, the tobacco plant with enhanced NUE contains lower amounts of at least three metabolites in at least five tissues. In a further aspect, the tobacco plant with enhanced NUE contains lower amounts of at least four metabolites in at least five tissues. In a further aspect, the tobacco plant with enhanced NUE contains lower amounts of at least five metabolites in at least five tissues.

[0190] In another aspect of the methods provided herein, a tobacco plant with enhanced NUE comprises reduced NUE in at least one tissue compared to a tobacco line that comprises a higher amount of at least one metabolite. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least one metabolite in two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises higher amounts of at least four metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least five metabolites in at least two tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least one metabolite in three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least two metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least three metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least four metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least five metabolites in at least three tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least one metabolite in four tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least two metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least three metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least four metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least five metabolites in at least four tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least one metabolite in five tissues. In a further aspect, tobacco plants with enhanced NUE contain elevated amounts of at least two metabolites in at least five tissues.In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least three metabolites in at least five tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least four metabolites in at least five tissues. In a further aspect, tobacco plants with enhanced NUE contain higher amounts of at least five metabolites in at least five tissues.

[0191] In another aspect of the methods provided herein, a tobacco plant with enhanced NUE comprises reduced NUE in at least one tissue compared to a tobacco line comprising an equivalent amount of at least one metabolite. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least two metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least three metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least four metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least five metabolites in at least one tissue. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least one metabolite in two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least two metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least three metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE comprises an equivalent amount of at least four metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least five metabolites in at least two tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least one metabolite in three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least two metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least three metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least four metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least five metabolites in at least three tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least one metabolite in four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least two metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least three metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least four metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least five metabolites in at least four tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least one metabolite in five tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least two metabolites in at least five tissues. In a further aspect, a tobacco plant with enhanced NUE contains equal amounts of at least three metabolites in at least five tissues.In a further aspect, the tobacco plant with enhanced NUE comprises equal amounts of at least four metabolites in at least five tissues. In a further aspect, the tobacco plant with enhanced NUE comprises equal amounts of at least five metabolites in at least five tissues.

[0192] In another aspect, the methods provided herein comprise determining the amount of a metabolite using a method selected from the group consisting of liquid chromatography / mass spectrometry (LC / MS), high performance liquid chromatography (HPLC), ultra HPLC (UHPLC), mass spectrometry (MS), tandem mass spectrometry (MS / MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), X-ray fluorescence spectroscopy (XRF), ion chromatography (IC), gas chromatography (GC), gas chromatography / mass spectrometry (GC / MS), capillary electrophoresis / mass spectrometry (CE-MS), ion mobility spectrometry / mass spectrometry (IMS / MS), X-ray diffraction, nuclear magnetic resonance (NMR), emission spectrometry, polarography, ultraviolet-visible spectroscopy, infrared spectroscopy, and thin layer chromatography.

[0193] In one aspect, the present disclosure provides and encompasses a method for determining the NUE of a tobacco line using a metabolite profile, comprising isolating a metabolite profile from a tobacco plant of the tobacco line, determining the amount of each metabolite comprising the metabolite profile, and determining the NUE of the tobacco line by comparing the metabolite profile to a control metabolite profile from a control tobacco line comprising known NUE. In a further aspect of the method, the NUE comprises enhanced NUE compared to the control tobacco line. In another aspect of the method, the metabolite profile is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue.

[0194] In one aspect of the methods provided herein, a metabolite signature comprises at least two metabolites. In a further aspect, a metabolite signature comprises at least three metabolites. In a further aspect, a metabolite signature comprises at least four metabolites. In a further aspect, a metabolite signature comprises at least five metabolites. In a further aspect, a metabolite signature comprises at least six metabolites. In a further aspect, a metabolite signature comprises at least seven metabolites. In a further aspect, a metabolite signature comprises at least eight metabolites. In a further aspect, a metabolite signature comprises at least nine metabolites. In a further aspect, a metabolite signature comprises at least ten metabolites. In a further aspect, a metabolite signature comprises at least eleven metabolites. In a further aspect, a metabolite signature comprises at least twelve metabolites. In a further aspect, a metabolite signature comprises at least thirteen metabolites. In a further aspect, a metabolite signature comprises at least fourteen metabolites. In a further aspect, a metabolite signature comprises at least fifteen metabolites. In a further aspect, a metabolite signature comprises at least twenty metabolites. In a further aspect, a metabolite signature comprises at least twenty-five metabolites. In a further aspect, the metabolite signature comprises at least thirty metabolites. In a further aspect, the metabolite signature comprises at least thirty-five metabolites. In a further aspect, the metabolite signature comprises at least forty metabolites. In a further aspect, the metabolite signature comprises at least forty-five metabolites. In a further aspect, the metabolite signature comprises at least fifty metabolites. In a further aspect, the metabolite signature comprises two to fifty metabolites. In a further aspect, the metabolite signature comprises three to forty-five metabolites. In a further aspect, the metabolite signature comprises three to forty metabolites. In a further aspect, the metabolite signature comprises four to thirty-five metabolites. In a further aspect, the metabolite signature comprises five to thirty metabolites. In a further aspect, the metabolite signature comprises six to twenty-five metabolites. In a further aspect, the metabolite signature comprises seven to twenty metabolites. In a further aspect, the metabolite signature comprises eight to fifteen metabolites. In a further aspect, the metabolite signature comprises nine to fourteen metabolites. In a further aspect, the metabolite signature comprises ten to thirteen metabolites. In a further aspect, the metabolite signature comprises ten to twelve metabolites.

[0195] In one aspect, the present disclosure provides and includes a method for breeding a tobacco line comprising a metabolite profile associated with enhanced NUE, comprising determining the metabolite profile of a first tobacco plant from a first tobacco line, wherein the first tobacco plant comprises enhanced NUE compared to a control tobacco plant lacking the metabolite profile, hybridizing the first plant with a second plant from a second tobacco line, and obtaining at least one progeny seed from the hybridization, wherein a progeny plant grown from the at least one progeny seed comprises the metabolite profile, and wherein the progeny plant comprises enhanced NUE compared to a control plant lacking the metabolite profile. In a further aspect of the method, the progeny plant is hybridized with a third plant from the first tobacco line. In another aspect, the first tobacco line is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925. On the other hand, the second tobacco line is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. On the other hand, the metabolite profile comprises a leaf metabolite profile. On the other hand, the metabolite profile comprises a root metabolite profile. On the other hand, compared with the metabolite profile of the control tobacco plant, the metabolite profile comprises 4-guanidinobutyrate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof, of a higher amount. In another aspect, the metabolite signature comprises lower amounts of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, naringenin, or any combination thereof, as compared to the metabolite signature of a control tobacco plant.

[0196] In another aspect, the methods provided herein comprise tobacco plants containing enhanced NUE, wherein the enhanced NUE comprises an increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE when grown under the same conditions. In a further aspect, the enhanced NUE comprises an increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE when grown under the same conditions. In a further aspect, the enhanced NUE comprises an increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE when grown under the same conditions. In a further aspect, the enhanced NUE comprises an increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE when grown under the same conditions. In a further aspect, the enhanced NUE comprises an increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE when grown under the same conditions.

[0197] In one aspect, the present specification provides and encompasses a method for selecting tobacco plants, comprising obtaining a tobacco plant population, isolating at least one metabolite associated with enhanced NUE from at least one tobacco plant from the tobacco plant population, and selecting at least one tobacco plant that contains a higher amount of the at least one metabolite compared to a control tobacco plant. In a further aspect of the method, the selected tobacco plant contains enhanced NUE compared to the control tobacco plant. In a further aspect of the method, the at least one metabolite is selected from the group consisting of 4-guanidinobutyrate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof. In a further aspect of the method, the metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue.

[0198] In one aspect, the present specification provides and encompasses a method for selecting a tobacco plant, comprising obtaining a population of tobacco plants, isolating at least one metabolite associated with enhanced NUE from at least one tobacco plant from the population of tobacco plants, and selecting at least one tobacco plant that comprises a lower amount of the at least one metabolite compared to a control tobacco plant. In a further aspect of the method, the selected tobacco plant comprises enhanced NUE compared to the control tobacco plant. In a further aspect of the method, at least one metabolite is selected from X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, naringenin, or any combination thereof. In a further aspect of the method, the metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, flower tissue, meristem tissue, and stem tissue.

[0199] In one aspect, the present specification provides and encompasses a method for screening a tobacco plant for a first metabolite signature associated with enhanced NUE, comprising isolating the first metabolite signature from the tobacco plant, determining the amount of at least one metabolite comprising the first metabolite signature, comparing the first metabolite signature to a second metabolite signature of a control tobacco plant comprising a known NUE, and determining whether the first metabolite signature is associated with enhanced NUE.

[0200] On the one hand, this specification provides and includes a kind of modified tobacco seed or the tobacco plant grown by it, it comprises the cis gene polynucleotide that contains the heterologous promoter that is operably connected to coding region, wherein compared with the unmodified control tobacco plant that lacks the cis gene polynucleotide when growing under the same conditions, described modified tobacco plant comprises enhanced nitrogen use efficiency.Further aspect, modified tobacco seed or tobacco plant comprise the heterologous promoter that is selected from lower group: constitutive promoter, inducible promoter, tissue preferred promoter and tissue specific promoter.On the other hand, heterologous promoter comprises the polynucleotide sequence from tobacco genome.On the other hand, heterologous promoter comprises the polynucleotide sequence from plant genome.On the other hand, tissue preferred promoter is leaf preferred promoter.On the other hand, tissue preferred promoter is root preferred promoter.Further aspect, modified tobacco seed or tobacco plant are burley tobacco varieties.

[0201] In a further aspect, compared with the unmodified tobacco plant that lacks the cis gene polynucleotide when growing under the same conditions, the modified tobacco seed of the present invention or tobacco plant comprises a lower amount of TSNA. In a further aspect, compared with the unmodified tobacco plant that lacks the cis gene polynucleotide when growing under the same conditions, the modified tobacco seed or tobacco plant comprises a lower amount of N'-nitrosonenornicotine (NNN). In a further aspect, compared with the unmodified tobacco plant that lacks the cis gene polynucleotide when growing under the same conditions, the modified tobacco seed or tobacco plant comprises a lower amount of 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK). In a further aspect, compared with the unmodified tobacco plant that lacks the cis gene polynucleotide when growing under the same conditions, the modified tobacco seed or tobacco plant comprises a lower amount of N'-nitrosoanatabine (NAT). In a further aspect, compared with the unmodified tobacco plant that lacks the cis gene polynucleotide when growing under the same conditions, the modified tobacco seed or tobacco plant comprises a lower amount of N'-nitrosoanabasine (NAB). In a further aspect, compared with the unmodified tobacco plant that lacks the cis-gene polynucleotide when growing under the same conditions, modified tobacco seed or tobacco plant comprise the alkaloid of lower amount. In a further aspect, compared with the unmodified tobacco plant that lacks the cis-gene polynucleotide when growing under the same conditions, modified tobacco seed or tobacco plant comprise the nicotine of lower amount. In a further aspect, compared with the unmodified tobacco plant that lacks the cis-gene polynucleotide when growing under the same conditions, modified tobacco seed or tobacco plant comprise the nornicotine of lower amount. In a further aspect, compared with the unmodified tobacco plant that lacks the cis-gene polynucleotide when growing under the same conditions, modified tobacco seed or tobacco plant comprise the anabasine of lower amount. In a further aspect, compared with the unmodified tobacco plant that lacks the cis-gene polynucleotide when growing under the same conditions, modified tobacco seed or tobacco plant comprise the anatabine of lower amount.

[0202] In a further aspect, the modified tobacco seed or tobacco plant of the present specification comprises a coding region encoding a polypeptide that is at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 75% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 80% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 85% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 90% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 95% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 96% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 97% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 98% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 99% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-8.

[0203] In a further aspect, the modified tobacco seed or tobacco plant of the present specification comprises a coding region encoding a polypeptide that is at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 75% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 80% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 85% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 90% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 95% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 96% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 97% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 98% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is at least 99% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 9-16. In a further aspect, the modified tobacco seed or tobacco plant comprises a coding region encoding a polypeptide that is identical to a sequence selected from the group consisting of SEQ ID NOs: 9-16.

[0204] In a further aspect, the modified tobacco seed or tobacco plant comprises a leaf-preferred promoter encoded by a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof. In a further aspect, the leaf-preferred promoter is encoded by a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof.

[0205] In a further aspect, the modified tobacco seed or tobacco plant comprises a root-preferred promoter encoded by a sequence that is at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof. In a further aspect, the root-preferred promoter is encoded by a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof.

[0206] In a further aspect, the modified tobacco plant of the present disclosure comprises higher levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0207] In a further aspect, the modified tobacco plant of the present disclosure comprises higher levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, X-23454, X-23580, and X-23852 in leaf tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0208] In a further aspect, the modified tobacco plant of the present disclosure comprises lower levels of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0209] In a further aspect, the modified tobacco plant of the present disclosure comprises lower levels of a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin in leaf tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0210] In one aspect, the present specification provides and encompasses a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 75% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 80% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 85% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 90% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide at least 95% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 96% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 97% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 98% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is at least 99% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a polypeptide that is 100% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0211] In one aspect, the present specification provides and encompasses a cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising a cis gene polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the modified tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0212] In one aspect, the present disclosure provides and includes greenhouses, growth chambers, or fields comprising modified tobacco seeds or plants disclosed herein. In one aspect, the present disclosure provides and includes methods of growing the tobacco plants of the present disclosure in greenhouses, growth chambers, or fields.

[0213] On the one hand, this specification sheets provides and comprises modified tobacco seed or the tobacco plant by its growth, it comprises at least one sudden change in the endogenous gene seat of the polypeptide that coding is selected from down group: SEQ ID NOs:25-40, wherein with the unmodified control tobacco plant that at least lacks at least one sudden change when growing under the same conditions, compare, modified tobacco seed or tobacco plant comprise the nitrogen utilization efficiency of enhancement.In further aspect, the sudden change in the endogenous gene seat is selected from down group: insertion, disappearance, replacement and inversion.On the other hand, the sudden change in the endogenous gene seat is silent mutation, non-silent mutation or null mutation.In further aspect, modified tobacco seed or modified tobacco plant are burley tobacco varieties.

[0214] In a further aspect, compared with the unmodified tobacco plant that grows under the same conditions, the modified tobacco plant comprises a higher level of metabolite that is selected from the group consisting of: 4-guanidinobutyrate, syringaldehyde, thiamine and p-hydroxybenzaldehyde in root tissue. In a further aspect, compared with the unmodified tobacco plant that grows under the same conditions, the modified tobacco plant comprises a higher level of metabolite that is selected from the group consisting of: 4-guanidinobutyrate, X-23454, X-23580 and X-23852 in leaf tissue. In a further aspect, compared with the unmodified tobacco plant that grows under the same conditions, the modified tobacco plant comprises a lower level of metabolite that is selected from the group consisting of: X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutaric acid, X-23937, X-23916 and 1-methyladenine in root tissue. In a further aspect, the modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin in leaf tissue compared to an unmodified tobacco plant grown under the same conditions.

[0215] In one aspect, the present disclosure provides and encompasses a recombinant DNA construct comprising a heterologous promoter operably linked to a guide RNA comprising at least 18 consecutive nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 19 consecutive nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 20 consecutive nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 21 consecutive nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 22 consecutive nucleotides identical to or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 23 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 24 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 25 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 26 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 27 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the guide RNA comprises at least 28 consecutive nucleotides that are 100% identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40.

[0216] In one aspect, the present specification provides and includes cured tobacco materials or tobacco products comprising the cured tobacco materials, wherein the cured tobacco materials are made from a tobacco plant comprising at least one mutation in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises enhanced NUE compared to an unmodified control tobacco plant lacking the at least one mutation when grown under the same conditions. In a further aspect, the tobacco plant comprises at least two mutations in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least three mutations in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least four mutations in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least five mutations in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40. In a further aspect, the tobacco plant comprises at least six mutations in the endogenous locus of the polypeptide that is selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least seven mutations in the endogenous locus of the polypeptide that is selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least eight mutations in the endogenous locus of the polypeptide that is selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least nine mutations in the endogenous locus of the polypeptide that is selected from the group consisting of SEQ ID NOs:25-40. In a further aspect, the tobacco plant comprises at least ten mutations in the endogenous locus of the polypeptide that is selected from the group consisting of SEQ ID NOs:25-40.

[0217] In one aspect, the present disclosure provides and encompasses a modified tobacco seed or tobacco plant grown therefrom, comprising a cisgene polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises enhanced NUE compared to an unmodified control tobacco plant lacking the cisgene polynucleotide when grown under the same conditions. In a further aspect, the cisgene polynucleotide comprises a polynucleotide encoding an sRNA at least 90% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgene polynucleotide comprises a polynucleotide encoding an sRNA at least 91% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cisgene polynucleotide comprises a polynucleotide encoding an sRNA at least 92% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 93% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 94% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 95% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 96% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 97% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 98% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is at least 99% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide comprises a polynucleotide encoding a sRNA that is 100% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter. In a further aspect, the tissue-preferred promoter is a leaf-preferred promoter.In a further aspect, the tissue-preferred promoter is a root-preferred promoter.

[0218] In a further aspect, the sRNA comprises at least 18 nucleotides. In a further aspect, the sRNA comprises at least 19 nucleotides. In a further aspect, the sRNA comprises at least 20 nucleotides. In a further aspect, the sRNA comprises at least 21 nucleotides. In a further aspect, the sRNA comprises at least 22 nucleotides. In a further aspect, the sRNA comprises at least 23 nucleotides. In a further aspect, the sRNA comprises at least 24 nucleotides. In a further aspect, the sRNA comprises at least 25 nucleotides. In a further aspect, the sRNA comprises at least 26 nucleotides. In a further aspect, the sRNA comprises at least 27 nucleotides. In a further aspect, the sRNA comprises at least 28 nucleotides. In a further aspect, the sRNA is selected from the group consisting of a microRNA, a small interfering RNA (siRNA), a trans-acting siRNA, and precursors thereof. In a further aspect, the sRNA downregulates expression or translation of a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0219] In one aspect, the present disclosure provides and encompasses a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 90% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 91% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 92% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 93% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 94% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 95% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 96% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 97% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 98% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding an sRNA that is at least 99% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the recombinant DNA construct comprises a polynucleotide encoding a sRNA that is 100% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0220] In one aspect, the present specification provides and encompasses a cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising a cis gene polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding an sRNA at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, and wherein the modified tobacco seed or tobacco plant comprises enhanced NUE compared to an unmodified control tobacco plant lacking the cis gene polynucleotide when grown under the same conditions. In a further aspect, the cis gene polynucleotide encodes an sRNA at least 90% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA at least 91% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA at least 92% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 93% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 94% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 95% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 96% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 97% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 98% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is at least 99% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56. In a further aspect, the cis gene polynucleotide encodes an sRNA that is 100% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0221] In one aspect, the present disclosure provides and encompasses a method for increasing NUE in a tobacco plant, comprising introducing a cis-gene nucleic acid molecule into a tobacco cell, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises increased NUE compared to a tobacco plant lacking the cis-gene nucleic acid molecule. In another aspect, the method further comprises crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0222] In one aspect, the present specification provides and encompasses a method for enhancing NUE in a tobacco plant, comprising introducing a modification of a nucleic acid molecule encoding a gene having a sequence selected from SEQ ID NOs: 41-56 into a tobacco cell, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the modification. In another aspect, the method further comprises crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant. In another aspect, the modification is introduced by a method comprising using an RNA-guided nuclease. In another aspect, the RNA-guided nuclease is selected from the group consisting of Cas9 nuclease, Cpf1 nuclease, CasX nuclease, CasY nuclease, and functional homologs thereof. In another aspect, the modification is selected from the group consisting of an insertion, a substitution, an inversion, and a deletion.

[0223] In one aspect, the present disclosure provides and encompasses a method for enhancing NUE in a tobacco plant, comprising introducing into a tobacco cell a nucleic acid encoding a small RNA (sRNA) homologous to at least 18 contiguous nucleic acids encoding a nucleic acid molecule encoding a gene having a sequence selected from SEQ ID NOs: 41-56, and regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the sRNA. In another aspect, the method further comprises crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant. In a further aspect, the method comprises introducing an sRNA selected from the group consisting of a microRNA, a small interfering RNA (siRNA), a trans-acting siRNA, and precursors thereof.

[0224] In one aspect, the present specification provides and includes a method comprising providing a first tobacco plant population comprising an enhanced NUE, genotyping the first tobacco plant population for the presence or absence of a molecular marker within 20 cM of the enhanced NUE locus; and selecting one or more tobacco plants that have been genotyped and found to contain the molecular marker. In a further aspect, the method disclosed herein comprises genotyping the first tobacco plant population for the presence or absence of a molecular marker within 15 cM of the enhanced NUE locus. In a further aspect, the method disclosed herein comprises genotyping the first tobacco plant population for the presence or absence of a molecular marker within 10 cM of the enhanced NUE locus. In a further aspect, the method disclosed herein comprises genotyping the first tobacco plant population for the presence or absence of a molecular marker within 9 cM of the enhanced NUE locus. In a further aspect, the method disclosed herein comprises genotyping the first tobacco plant population for the presence or absence of a molecular marker within 8 cM of the enhanced NUE locus. In a further aspect, the method disclosed herein comprises genotyping the first tobacco plant population for the presence or absence of a molecular marker within 7 cM of the enhanced NUE locus. In a further aspect, the methods disclosed herein include genotyping the first tobacco plant population for the presence or absence of a molecular marker within 6 cM of the enhanced NUE locus. In a further aspect, the methods disclosed herein include genotyping the first tobacco plant population for the presence or absence of a molecular marker within 5 cM of the enhanced NUE locus. In a further aspect, the methods disclosed herein include genotyping the first tobacco plant population for the presence or absence of a molecular marker within 4 cM of the enhanced NUE locus. In a further aspect, the methods disclosed herein include genotyping the first tobacco plant population for the presence or absence of a molecular marker within 3 cM of the enhanced NUE locus. In a further aspect, the methods disclosed herein include genotyping the first tobacco plant population for the presence or absence of a molecular marker within 2 cM of the enhanced NUE locus. In a further aspect, the methods disclosed herein include genotyping the first tobacco plant population for the presence or absence of a molecular marker within 1 cM of the enhanced NUE locus. In a further aspect, the method disclosed herein comprises genotyping a first tobacco plant population for the presence of a molecular marker within 0.5 cM of an enhanced NUE locus. In a further aspect, the method comprises hybridizing one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seeds from the hybridization. In a further aspect, the molecular marker is selected from the group consisting of a SNP marker, an INDEL marker, a RFLP marker, a SSR marker, an AFLP marker, and a RAPD marker.

[0225] In a further aspect, the methods provided herein include a tobacco plant comprising an enhanced NUE locus, the locus comprising a polynucleotide encoding a polypeptide at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide at least 75% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide at least 80% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide at least 85% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide at least 90% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide at least 95% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide at least 96% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 97% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 98% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is at least 99% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the polynucleotide encodes a polypeptide that is 100% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8. In a further aspect, the enhanced NUE locus is genetically linked to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 57-64. In another aspect, the enhanced NUE locus is genetically linked to the G at position 57 of SEQ ID NO: 58. In another aspect, the enhanced NUE locus is genetically linked to the C at position 117 of SEQ ID NO: 58. In another aspect, the enhanced NUE locus is genetically linked to the G at position 57 and the C at position 117 of SEQ ID NO: 58. In another aspect, the enhanced NUE locus is genetically linked to the T nucleotide at position 147 of SEQ ID NO: 57. In another aspect, the enhanced NUE locus is genetically linked to the G nucleotide at position 162 of SEQ ID NO: 59. In another aspect, the enhanced NUE locus is genetically linked to the C nucleotide at position 36 of SEQ ID NO: 60. In another aspect, the enhanced NUE locus is genetically linked to the T nucleotide at position 36 of SEQ ID NO: 61. In another aspect, the enhanced NUE locus is genetically linked to the T nucleotide at position 36 of SEQ ID NO: 62.In another aspect, the enhanced NUE locus is genetically linked to the G nucleotide at position 36 of SEQ ID NO: 63. In another aspect, the enhanced NUE locus is genetically linked to the T nucleotide at position 36 of SEQ ID NO: 64.

[0226] In the further aspect of the method that this paper provides, the first tobacco plant colony is the Maryland kind.In further aspect, the method that this paper provides comprises the first tobacco plant colony that is selected from lower group: MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.In further aspect, the method that this paper provides comprises the second tobacco plant colony that is selected from lower group: TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0227] In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker. In a further aspect, the methods provided herein include progeny seeds comprising the enhanced NUE. In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker within 20 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker within 15 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker within 10 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker within 9 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker within 8 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising the molecular marker within 7 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 6 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 5 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 4 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 3 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 2 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 1 cM of the enhanced NUE efficiency locus provided herein. In a further aspect, the methods provided herein include progeny seeds comprising a molecular marker within 0.5 cM of the enhanced NUE efficiency locus provided herein.

[0228] In one aspect, the present specification provides and includes a method comprising providing a first tobacco plant population, genotyping the first tobacco plant population for the presence of an enhanced NUE allele at a locus encoded by a sequence selected from the group consisting of SEQ ID NOs: 9-16; and selecting one or more genotyped tobacco plants comprising the enhanced NUE allele. In a further aspect, the method further comprises hybridizing the one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seeds from the hybridization.

[0229] In one aspect, the present specification provides and includes a method for introgressing an enhanced NUE trait into a tobacco variety, comprising crossing a first tobacco variety comprising an enhanced nitrogen use efficiency trait with a second tobacco variety lacking the enhanced nitrogen use efficiency trait, obtaining progeny seeds from the cross, genotyping at least one progeny seed for a molecular marker associated with the enhanced nitrogen use efficiency trait, wherein the molecular marker is located within 20 cM of a locus having a sequence selected from the group consisting of SEQ ID NOs: 9-16, and selecting progeny seeds comprising the enhanced nitrogen use efficiency trait.

[0230] In one aspect, the present disclosure provides and encompasses a method for selecting tobacco plants having an enhanced NUE trait, comprising isolating nucleic acid from a tobacco germplasm collection, assaying the isolated nucleic acid for one or more markers located within 20 cM of a locus having a sequence selected from the group consisting of SEQ ID NOs: 9-16, and selecting tobacco plants comprising the enhanced NUE trait. In a further aspect, the method further comprises hybridizing one or more selected tobacco plants with a second tobacco plant; and obtaining progeny seeds from the hybrid.

[0231] In one aspect, the present specification provides and encompasses a method for selecting tobacco plants having an enhanced NUE trait, comprising isolating nucleic acid from a tobacco germplasm collection, assaying the isolated nucleic acid for one or more markers within 20 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64, and selecting tobacco plants comprising the enhanced NUE trait. In a further aspect, the method disclosed herein comprises assaying the isolated nucleic acid for one or more markers within 15 cM of a marker selected from the group consisting of SEQ ID NOs: 58. In a further aspect, the method disclosed herein comprises assaying the isolated nucleic acid for one or more markers within 10 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the method disclosed herein comprises assaying the isolated nucleic acid for one or more markers within 9 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the method disclosed herein comprises assaying the isolated nucleic acid for one or more markers within 8 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers within 7 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers within 6 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers within 5 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers within 4 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers within 3 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers within 2 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers located within 1 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers located within 0.5 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64. In a further aspect, the methods disclosed herein comprise assaying an isolated nucleic acid for one or more markers located within 0.5 cM of a marker selected from the group consisting of SEQ ID NOs: 57-64.In another aspect, the allele associated with enhanced NUE comprises a G nucleotide at position 57 of SEQ ID NO:58. In another aspect, the allele associated with enhanced NUE comprises a C nucleotide at position 117 of SEQ ID NO:58. In another aspect, the allele associated with enhanced NUE comprises a G nucleotide at position 57 of SEQ ID NO:58 and a C nucleotide at position 117 of SEQ ID NO:58. In another aspect, the allele associated with enhanced NUE comprises a T nucleotide at position 147 of SEQ ID NO:57. In another aspect, the allele associated with enhanced NUE comprises a G nucleotide at position 162 of SEQ ID NO:59. In another aspect, the allele associated with enhanced NUE comprises a C nucleotide at position 36 of SEQ ID NO:60. In another aspect, the allele associated with enhanced NUE comprises a T nucleotide at position 36 of SEQ ID NO:61. In another aspect, the allele associated with enhanced NUE comprises a T nucleotide at position 36 of SEQ ID NO:62. In another aspect, the allele associated with enhanced NUE comprises a G nucleotide at position 36 of SEQ ID NO: 63. In another aspect, the allele associated with enhanced NUE comprises a T nucleotide at position 36 of SEQ ID NO: 64. In a further aspect, tobacco plants can be selected that comprise any combination of alleles associated with enhanced NUE disclosed herein.

[0232] The following are exemplary implementations.

[0233] Embodiment 1. A method for determining nitrogen utilization efficiency (NUE) of a tobacco line, comprising:

[0234] a. obtaining at least one metabolite from a tobacco plant of said tobacco line;

[0235] b. determining the amount of the at least one metabolite; and

[0236] C. Determining the nitrogen utilization efficiency of the tobacco line based on the amount of the at least one metabolite identified in step (b).

[0237] Embodiment 2. The method of embodiment 1, wherein the at least one metabolite is obtained from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue.

[0238] Embodiment 3. The method of embodiment 1 or 2, wherein the plant tissue comprises leaf tissue.

[0239] Embodiment 4. The method of any one of embodiments 1-3, wherein the plant tissue comprises root tissue.

[0240] Embodiment 5. The method of any one of embodiments 1-4, wherein the at least one metabolite is selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, D-23937, X-23937, X-23916, 1-methyladenine, 4-guanidinobutyrate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, X-23366, N-acetylphenylalanine, naringenin, X-23454, X-23580, and X-23852.

[0241] Embodiment 6. The method of any one of embodiments 1-5, wherein the NUE comprises enhanced NUE compared to a tobacco line comprising a lower amount of the at least one metabolite in the at least one tissue.

[0242] Embodiment 7. The method of any one of embodiments 1-6, wherein the NUE comprises enhanced NUE compared to a tobacco line comprising an equivalent amount of the at least one metabolite in the at least one tissue.

[0243] Embodiment 8. The method of any one of embodiments 1-7, wherein the NUE comprises enhanced NUE compared to a tobacco line comprising an equivalent amount of the at least one metabolite in the at least one tissue.

[0244] Embodiment 9. The method of any one of embodiments 1-8, wherein the NUE comprises reduced NUE compared to a tobacco line comprising a lower amount of the at least one metabolite in the at least one tissue.

[0245] Embodiment 10. The method of any one of embodiments 1-9, wherein the NUE comprises reduced NUE compared to a tobacco line comprising an equivalent amount of the at least one metabolite in the at least one tissue.

[0246] Embodiment 11. The method of any one of embodiments 1-10, wherein the NUE comprises reduced NUE compared to a tobacco line comprising an equivalent amount of the at least one metabolite in the at least one tissue.

[0247] Embodiment 12. A method according to any one of embodiments 1-11, wherein the determining the amount of the at least one metabolite comprises a method selected from the group consisting of liquid chromatography / mass spectrometry (LC / MS), high performance liquid chromatography (HPLC), ultra HPLC (UHPLC), mass spectrometry (MS), tandem mass spectrometry (MS / MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), X-ray fluorescence spectroscopy (XRF), ion chromatography (IC), gas chromatography (GC), gas chromatography / mass spectrometry (GC / MS), capillary electrophoresis / mass spectrometry (CE-MS), ion mobility spectrometry / mass spectrometry (IMS / MS), X-ray diffraction, nuclear magnetic resonance (NMR), emission spectrometry, polarography, ultraviolet-visible spectroscopy, infrared spectroscopy, and thin layer chromatography.

[0248] Embodiment 13. The method of any one of embodiments 1-12, wherein the at least one metabolite comprises at least two metabolites.

[0249] Embodiment 14. The method of any one of embodiments 1-13, wherein the at least one metabolite comprises at least five metabolites.

[0250] Embodiment 15. The method of any one of embodiments 1-14, wherein the at least one metabolite comprises at least ten metabolites.

[0251] Embodiment 16. A method for determining nitrogen utilization efficiency (NUE) of a tobacco line using metabolite profiling, comprising:

[0252] a. isolating the metabolite profile from tobacco plants of the tobacco line;

[0253] b. determining the amount of each metabolite comprising the metabolite signature; and

[0254] c. Determining the NUE of the tobacco line by comparing the metabolite signature to a control metabolite signature from a control tobacco line comprising a known NUE.

[0255] Embodiment 17. The method of embodiment 16, wherein the metabolite signature comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 metabolites.

[0256] Embodiment 18. The method of embodiment 16 or 17, wherein the NUE comprises enhanced NUE compared to the control tobacco line.

[0257] Embodiment 19. The method of any one of embodiments 16-18, wherein the NUE comprises reduced NUE compared to the control tobacco line.

[0258] Embodiment 20. The method of any one of embodiments 16-19, wherein the metabolite signature is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue.

[0259] Embodiment 21. A method of breeding a tobacco line comprising a metabolite profile associated with enhanced nitrogen utilization efficiency (NUE), comprising:

[0260] a. Determining a metabolite profile of a first tobacco plant from a first tobacco line, wherein the first tobacco plant comprises enhanced NUE compared to a control tobacco plant lacking the metabolite profile;

[0261] b. hybridizing said first tobacco plant with a second plant of a second tobacco line; and

[0262] c. obtaining at least one progeny seed from the cross of step (a), wherein a progeny plant grown from the at least one progeny seed comprises the metabolite signature, and wherein the progeny plant comprises enhanced NUE compared to a control plant lacking the metabolite signature.

[0263] Embodiment 22. The method of embodiment 21, further comprising:

[0264] d. Crossing the progeny plant with a tobacco plant from the first tobacco line.

[0265] Embodiment 23. The method of embodiment 21 or 22, wherein the first tobacco line is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925.

[0266] Embodiment 24. The method of any one of embodiments 21-23, wherein the second tobacco line is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0267] Embodiment 25. The method of any one of embodiments 21-24, wherein the metabolite signature comprises a leaf metabolite signature.

[0268] Embodiment 26. The method of any one of embodiments 21-25, wherein the metabolite signature comprises a root metabolite signature.

[0269] Embodiment 27. The method of any one of embodiments 21-26, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0270] Embodiment 28. The method of any one of embodiments 21-27, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0271] Embodiment 29. The method of any one of embodiments 21-28, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0272] Embodiment 30. The method of any one of embodiments 21-29, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0273] Embodiment 31. The method of any one of embodiments 21-30, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0274] Embodiment 32. The method of any one of embodiments 21-31, wherein the metabolite signature comprises equivalent amounts of 4-guanidinobutyrate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof, as compared to the metabolite signature of the control tobacco plant.

[0275] Embodiment 33. The method of any one of embodiments 21-32, wherein the metabolite signature comprises lower amounts of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, naringenin, or any combination thereof, as compared to the metabolite signature of the control tobacco plant.

[0276] Embodiment 34. A method according to any one of embodiments 21-33, wherein the metabolite signature comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 metabolites.

[0277] Embodiment 35. A method of selecting a tobacco plant, comprising:

[0278] a. Obtaining a tobacco plant population;

[0279] b. isolating at least one metabolite associated with enhanced nitrogen utilization efficiency (NUE) from at least one tobacco plant of said tobacco plant population; and

[0280] c. Selecting at least one tobacco plant that comprises an equivalent amount of the at least one metabolite as compared to a control tobacco plant.

[0281] Embodiment 36. The method of embodiment 35, wherein the tobacco plant selected in step (c) comprises equivalent NUE compared to the control tobacco plant.

[0282] Embodiment 37. The method of embodiment 35 or 36, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0283] Embodiment 38. The method of any one of embodiments 35-37, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0284] Embodiment 39. The method of any one of embodiments 35-38, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0285] Embodiment 40. The method of any one of embodiments 35-39, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0286] Embodiment 41. The method of any one of embodiments 35-40, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0287] Embodiment 42. The method of any one of embodiments 35-41, wherein the at least one metabolite is selected from the group consisting of 4-guanidinobutyrate, syringaldehyde, thiamine, p-hydroxybenzaldehyde, X-23454, X-23580, X-23852, or any combination thereof.

[0288] Embodiment 43. The method of any one of embodiments 35-42, wherein the at least one metabolite is isolated from leaf tissue or root tissue.

[0289] Embodiment 44. The method of any one of embodiments 35-43, wherein the at least one metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue.

[0290] Embodiment 45. A method of selecting a tobacco plant, comprising:

[0291] a. Obtaining a tobacco plant population;

[0292] b. isolating at least one metabolite associated with enhanced nitrogen utilization efficiency (NUE) from at least one tobacco plant of said tobacco plant population; and

[0293] c. Selecting at least one tobacco plant that comprises a lower amount of the at least one metabolite compared to a control tobacco plant.

[0294] Embodiment 46. The method of embodiment 45, wherein the tobacco plants selected in step (c) comprise equivalent NUE compared to control tobacco plants.

[0295] Embodiment 47. The method of embodiment 45 or 46, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant grown under the same conditions lacking the enhanced NUE.

[0296] Embodiment 48. The method of any one of embodiments 45-47, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant grown under the same conditions lacking the enhanced NUE.

[0297] Embodiment 49. The method of any one of embodiments 45-48, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0298] Embodiment 50. The method of any one of embodiments 45-49, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0299] Embodiment 51. The method of any one of embodiments 45-50, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0300] Embodiment 52. The method of any one of embodiments 45-51, wherein the at least one metabolite is selected from X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, 1-methyladenine, X-23453, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, X-23366, N-acetylphenylalanine, naringenin, or any combination thereof.

[0301] Embodiment 53. The method of any one of embodiments 45-52, wherein the at least one metabolite is isolated from leaf tissue or root tissue.

[0302] Embodiment 54. The method of any one of embodiments 45-53, wherein the at least one metabolite is isolated from a plant tissue selected from the group consisting of root tissue, leaf tissue, floral tissue, meristem tissue, and stem tissue.

[0303] Embodiment 55. A method of screening tobacco plants for a metabolite signature associated with enhanced nitrogen use efficiency (NUE), comprising:

[0304] a. isolating a first metabolite signature associated with enhanced NUE from the tobacco plant;

[0305] b. determining the amount of at least one metabolite comprising said first metabolite signature;

[0306] c. comparing the first metabolite signature to a second metabolite signature of a control tobacco plant comprising a known NUE; and

[0307] d. Determining whether the first metabolite signature is associated with enhanced NUE.

[0308] Embodiment 56. The method of embodiment 55, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0309] Embodiment 57. The method of embodiment 55 or 56, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0310] Embodiment 58. The method of any one of embodiments 55-57, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0311] Embodiment 59. The method of any one of embodiments 55-58, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0312] Embodiment 60. The method of any one of embodiments 55-59, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0313] Embodiment 61. A modified tobacco seed or a tobacco plant grown therefrom, comprising a cis-gene polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the modified tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis-gene polynucleotide when grown under the same conditions.

[0314] Embodiment 62. The modified tobacco seed or tobacco plant of embodiment 61, wherein the coding region encodes a polypeptide that is at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 1-8.

[0315] Embodiment 63. The modified tobacco seed or tobacco plant of embodiment 61 or 62, wherein the coding region comprises a polynucleotide sequence that is at least 70% identical or complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 9-16.

[0316] Embodiment 64. The modified tobacco seed or tobacco plant of any one of embodiments 61-63, wherein the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter.

[0317] Embodiment 65. The modified tobacco seed or tobacco plant of any one of embodiments 61-64, wherein the heterologous promoter comprises a polynucleotide sequence from the tobacco genome.

[0318] Embodiment 66. The modified tobacco seed or tobacco plant of any one of embodiments 61-65, wherein the heterologous promoter comprises a polynucleotide sequence from a plant genome.

[0319] Embodiment 67. The modified tobacco seed or tobacco plant of any one of embodiments 61-66, wherein the tissue-preferred promoter is a leaf-preferred promoter.

[0320] Embodiment 68. The modified tobacco seed or tobacco plant of any one of embodiments 61-67, wherein the tissue-preferred promoter is a root-preferred promoter.

[0321] Embodiment 69. The modified tobacco seed or tobacco plant of any one of embodiments 61-68, wherein the leaf-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof.

[0322] Embodiment 70. The modified tobacco seed or tobacco plant of any one of embodiments 61-69, wherein the root-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof.

[0323] Embodiment 71. The modified tobacco plant of any one of embodiments 61-70, wherein the modified tobacco plant comprises equivalent levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0324] Embodiment 72. The modified tobacco plant of any one of embodiments 61-71, wherein the modified tobacco plant comprises equivalent levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, X-23454, X-23580, and X-23852 in leaf tissue as compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0325] Embodiment 73. The modified tobacco plant of any one of embodiments 61-72, wherein the modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0326] Embodiment 74. The modified tobacco plant of any one of embodiments 61-73, wherein said modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin in leaf tissue compared to an unmodified tobacco plant lacking said cis gene polynucleotide when grown under identical conditions.

[0327] Embodiment 75. The modified tobacco seed or plant of any one of Embodiments 61-74, wherein the modified tobacco seed or plant is a Burley variety.

[0328] Embodiment 76. The modified tobacco seed or plant of any one of embodiments 61-75, wherein the modified tobacco seed or plant comprises a lower amount of TSNAs compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0329] Embodiment 77. A recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0330] Embodiment 78. A cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising a cis-gene polynucleotide comprising a heterologous promoter operably linked to a coding region, wherein the modified tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis-gene polynucleotide when grown under the same conditions.

[0331] Embodiment 79. A greenhouse, growth chamber, or field comprising the modified tobacco seed or plant of any one of embodiments 61-76.

[0332] Embodiment 80. A modified tobacco seed or tobacco plant grown therefrom, comprising at least one mutation in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the at least one mutation when grown under the same conditions.

[0333] Embodiment 81. The modified tobacco seed or tobacco plant of embodiment 80, wherein the at least one mutation is selected from the group consisting of an insertion, a deletion, a substitution, and an inversion.

[0334] Embodiment 82. The modified tobacco seed or tobacco plant of embodiment 80 or 81, wherein the at least one mutation is a null mutation.

[0335] Embodiment 83. The modified tobacco plant of any one of embodiments 80-82, wherein the modified tobacco plant comprises equivalent levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue as compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0336] Embodiment 84. The modified tobacco plant of any one of embodiments 80-83, wherein the modified tobacco plant comprises equivalent levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, X-23454, X-23580, and X-23852 in leaf tissue as compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0337] Embodiment 85. The modified tobacco plant of any one of embodiments 80-84, wherein the modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, alpha-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0338] Embodiment 86. The modified tobacco plant of any one of embodiments 80-85, wherein the modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin in leaf tissue compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0339] Embodiment 87. The modified tobacco seed or plant of any one of Embodiments 80-86, wherein the modified tobacco seed or plant is a Burley variety.

[0340] Embodiment 88. The modified tobacco seed or plant of any one of embodiments 80-87, wherein the modified tobacco seed or plant comprises a lower amount of TSNAs compared to an unmodified tobacco plant lacking the at least one mutation when grown under the same conditions.

[0341] Embodiment 89. A recombinant DNA construct comprising a heterologous promoter operably linked to a guide RNA, wherein the guide RNA comprises at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27 or at least 28 consecutive nucleotides that are identical or complementary to a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40.

[0342] Embodiment 90. A cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising at least one mutation in an endogenous locus encoding a polypeptide selected from the group consisting of SEQ ID NOs: 25-40, wherein the modified tobacco seed or tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the at least one mutation when grown under the same conditions.

[0343] Embodiment 91. A modified tobacco seed or tobacco plant grown therefrom, comprising a cis-gene polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA), said polynucleotide being at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56, wherein the modified tobacco seed or tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis-gene polynucleotide when grown under the same conditions.

[0344] Embodiment 92. The modified tobacco seed or tobacco plant of embodiment 91, wherein the sRNA comprises at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 nucleotides.

[0345] Embodiment 93. The modified tobacco seed or tobacco plant of embodiment 91 or 92, wherein the sRNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof.

[0346] Embodiment 94. The modified tobacco seed or tobacco plant of any one of embodiments 91-93, wherein the sRNA downregulates expression or translation of a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0347] Embodiment 95. The modified tobacco seed or tobacco plant of any one of embodiments 91-94, wherein the heterologous promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-preferred promoter, and a tissue-specific promoter.

[0348] Embodiment 96. The modified tobacco seed or tobacco plant of any one of embodiments 91-95, wherein the tissue-preferred promoter is a leaf-preferred promoter.

[0349] Embodiment 97. The modified tobacco seed or tobacco plant of any one of embodiments 91-96, wherein the tissue-preferred promoter is a root-preferred promoter.

[0350] Embodiment 98. The modified tobacco seed or tobacco plant of any one of embodiments 91-97, wherein the leaf-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 17-19, or a functional fragment thereof.

[0351] Embodiment 99. The modified tobacco seed or tobacco plant of any one of embodiments 91-98, wherein the root-preferred promoter is encoded by a sequence that is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 20-24, or a functional fragment thereof.

[0352] Embodiment 100. The modified tobacco seed or tobacco plant of any one of embodiments 91-99, wherein the heterologous promoter comprises a polynucleotide sequence from the tobacco genome.

[0353] Embodiment 101. The modified tobacco seed or tobacco plant of any one of embodiments 91-100, wherein the heterologous promoter comprises a polynucleotide sequence from a plant genome.

[0354] Embodiment 102. The modified tobacco plant of any one of embodiments 91-101, wherein the modified tobacco plant comprises equivalent levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, syringaldehyde, thiamine, and p-hydroxybenzaldehyde in root tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0355] Embodiment 103. The modified tobacco plant of any one of embodiments 91-102, wherein the modified tobacco plant comprises equivalent levels of a metabolite selected from the group consisting of 4-guanidinobutyrate, X-23454, X-23580, and X-23852 in leaf tissue as compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under identical conditions.

[0356] Embodiment 104. The modified tobacco plant of any one of embodiments 91-103, wherein the modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-2357, N-acetylmuramate, X-23319, X-23852, X-23330, α-ketoglutarate, X-21756, 4-hydroxy-2-ketoglutarate, X-23937, X-23916, and 1-methyladenine in root tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0357] Embodiment 105. The modified tobacco plant of any one of embodiments 91-104, wherein the modified tobacco plant comprises lower levels of a metabolite selected from the group consisting of X-23453, X-21756, X-11429, X-21796, N'-methylnicotinamide, cotinine, X-23389, N-acetylarginine, N-23366, N-acetylphenylalanine, and naringenin in leaf tissue compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0358] Embodiment 106. The modified tobacco seed or plant of any one of Embodiments 91-105, wherein the modified tobacco seed or plant is a Burley variety.

[0359] Embodiment 107. The modified tobacco seed or plant of any one of embodiments 91-106, wherein the modified tobacco seed or plant comprises a lower amount of TSNAs compared to an unmodified tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0360] Embodiment 108. A recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 41-56.

[0361] Embodiment 109. A cured tobacco material or a tobacco product comprising the cured tobacco material, wherein the cured tobacco material is made from a tobacco plant comprising a cis gene polynucleotide comprising a heterologous promoter operably linked to a polynucleotide encoding a small RNA (sRNA) that is at least 85% identical or complementary to a polynucleotide selected from SEQ ID NOs: 41-56, and wherein the modified tobacco seed or tobacco plant comprises enhanced nitrogen use efficiency compared to an unmodified control tobacco plant lacking the cis gene polynucleotide when grown under the same conditions.

[0362] Embodiment 110. A method of increasing nitrogen use efficiency (NUE) in a tobacco plant, comprising:

[0363] a. introducing a cis-gene nucleic acid molecule into tobacco cells; and

[0364] b. Regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the cis gene nucleic acid molecule.

[0365] Embodiment 111. The method of embodiment 110, further comprising:

[0366] c. crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0367] Embodiment 112. The method of embodiment 110 or 111, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant grown under the same conditions lacking the enhanced NUE.

[0368] Embodiment 113. The method of any one of embodiments 110-112, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0369] Embodiment 114. The method of any one of embodiments 110-113, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0370] Embodiment 115. The method of any one of embodiments 110-114, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0371] Embodiment 116. The method of any one of embodiments 110-115, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0372] Embodiment 117. A method of increasing nitrogen use efficiency (NUE) in a tobacco plant, comprising:

[0373] a. Modification of a nucleic acid molecule encoding a gene having a sequence selected from the group consisting of: SEQ ID NOs: 41-56; and

[0374] b. Regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the modification.

[0375] Embodiment 118. The method of claim 117, wherein the introducing comprises using an RNA-guided nuclease.

[0376] Embodiment 119. The method of claim 117 or 118, wherein the RNA-guided nuclease is selected from the group consisting of Cas9 nuclease, Cpf1 nuclease, CasX nuclease, CasY nuclease, and functional homologs thereof.

[0377] Embodiment 120. The method of any one of claims 117-119, wherein the modification is selected from the group consisting of an insertion, a substitution, an inversion, and a deletion.

[0378] Embodiment 121. The method of any one of claims 117-120, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0379] Embodiment 122. The method of any one of embodiments 117-121, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0380] Embodiment 123. The method of any one of embodiments 117-122, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0381] Embodiment 124. The method of any one of embodiments 117-123, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0382] Embodiment 125. The method of any one of embodiments 117-124, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0383] Embodiment 126. The method of any one of embodiments 117-125, further comprising:

[0384] c. crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0385] Embodiment 127. A method of increasing nitrogen use efficiency (NUE) in a tobacco plant, comprising:

[0386] a. introducing into tobacco cells a nucleic acid encoding a small RNA (sRNA), said nucleic acid being homologous to at least 18 consecutive nucleic acids encoding a gene having a sequence selected from the group consisting of: SEQ ID NOs: 41-56; and

[0387] b. Regenerating a modified tobacco plant from the tobacco cell, wherein the modified tobacco plant comprises enhanced NUE compared to a tobacco plant lacking the sRNA.

[0388] Embodiment 128. The method of embodiment 127, wherein the sRNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, and precursors thereof.

[0389] Embodiment 129. The method of claim 127 or 128, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0390] Embodiment 130. The method of any one of embodiments 127-129, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0391] Embodiment 131. The method of any one of embodiments 127-130, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0392] Embodiment 132. The method of any one of embodiments 127-131, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0393] Embodiment 133. The method of any one of embodiments 127-132, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0394] Embodiment 134. The method of any one of embodiments 127-133, wherein the method further comprises:

[0395] c. crossing the modified tobacco plant with a second tobacco plant or self-pollinating the modified tobacco plant.

[0396] Embodiment 135. A method comprising:

[0397] a. Providing a first tobacco plant population comprising enhanced nitrogen utilization efficiency;

[0398] b. Genotyping the first tobacco plant population for the presence of a molecular marker within 20 cM of an enhanced nitrogen use efficiency locus; and

[0399] c. selecting one or more tobacco plants comprising the molecular marker genotyped in step (b).

[0400] Embodiment 136. The method of embodiment 135, further comprising:

[0401] d. hybridizing one or more tobacco plants selected in step (c) with a second tobacco plant; and

[0402] e. Obtaining progeny seeds from the cross of step (d).

[0403] Embodiment 137. The method according to embodiment 135 or 136, wherein the molecular marker is selected from the group consisting of: SNP markers, INDEL markers, RFLP markers, SSR markers, AFLP markers and RAPD markers.

[0404] Embodiment 138. The method of any one of embodiments 135-137, wherein the enhanced nitrogen use efficiency locus comprises a polynucleotide encoding a polypeptide at least 70% identical or similar to a polypeptide selected from the group consisting of SEQ ID NOs: 1-8.

[0405] Embodiment 139. The method of any one of embodiments 135-138, wherein the enhanced nitrogen use efficiency locus comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 9-16.

[0406] Embodiment 140. The method according to any one of embodiments 135-139, wherein the molecular marker is selected from the group consisting of SEQ ID NOs: 57-64.

[0407] Embodiment 141. A method according to any one of embodiments 135-140, wherein the molecular marker comprises a G nucleotide at position 57 of SEQ ID NO:58.

[0408] Embodiment 142. A method according to any one of embodiments 135-141, wherein the molecular marker comprises a C nucleotide at position 117 of SEQ ID NO:58.

[0409] Embodiment 143. A method according to any one of embodiments 135-142, wherein the molecular marker comprises a G nucleotide at position 57 of SEQ ID NO:58 and a C nucleotide at position 117 of SEQ ID NO:58.

[0410] Embodiment 144. A method according to any one of embodiments 135-143, wherein the molecular marker comprises a T nucleotide at position 14 of SEQ ID NO:57.

[0411] Embodiment 145. A method according to any one of embodiments 135-144, wherein the molecular marker comprises a G nucleotide at position 162 of SEQ ID NO:59.

[0412] Embodiment 146. A method according to any one of embodiments 135-145, wherein the molecular marker comprises a C nucleotide at position 36 of SEQ ID NO:60.

[0413] Embodiment 147. A method according to any one of embodiments 135-146, wherein the molecular marker comprises a T nucleotide at position 36 of SEQ ID NO:61.

[0414] Embodiment 148. A method according to any one of embodiments 135-147, wherein the molecular marker comprises a T nucleotide at position 36 of SEQ ID NO:62.

[0415] Embodiment 149. A method according to any one of embodiments 135-148, wherein the molecular marker comprises a G nucleotide at position 36 of SEQ ID NO:63.

[0416] Embodiment 150. A method according to any one of embodiments 135-149, wherein the molecular marker comprises a T nucleotide at position 36 of SEQ ID NO:64.

[0417] Embodiment 151. The method of any one of embodiments 135-150, wherein the first population of tobacco plants is of the Maryland variety.

[0418] Embodiment 152. A method according to any one of embodiments 135-151, wherein the first tobacco plant population is a variety selected from the group consisting of: MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.

[0419] Embodiment 153. The method of any one of embodiments 135-152, wherein the second tobacco plant is a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0420] Embodiment 154. The method of any one of embodiments 135-153, wherein the progeny seeds comprise the molecular marker.

[0421] Embodiment 155. The method of any one of embodiments 135-154, wherein the progeny seeds comprise the enhanced nitrogen use efficiency.

[0422] Embodiment 156. A method according to any one of embodiments 135-155, wherein the molecular marker is within 15 cM of the enhanced nitrogen use efficiency locus.

[0423] Embodiment 157. A method according to any one of embodiments 135-156, wherein the molecular marker is within 10 cM of the enhanced nitrogen use efficiency locus.

[0424] Embodiment 158. A method according to any one of embodiments 135-157, wherein the molecular marker is within 5 cM of the enhanced nitrogen use efficiency locus.

[0425] Embodiment 159. A method according to any one of embodiments 135-158, wherein the molecular marker is within 2 cM of the enhanced nitrogen use efficiency locus.

[0426] Embodiment 160. The method of any one of embodiments 135-159, wherein the molecular marker is within 1 cM of the enhanced nitrogen use efficiency locus.

[0427] Embodiment 161. A method according to any one of embodiments 135-160, wherein the molecular marker is within 0.5 cM of the enhanced nitrogen use efficiency locus.

[0428] Embodiment 162. A method comprising:

[0429] a. Providing a first tobacco plant population;

[0430] b. genotyping the first tobacco plant population for the presence of an enhanced nitrogen use efficiency allele at a locus encoded by a sequence selected from the group consisting of SEQ ID NOs: 9-16; and

[0431] c. selecting one or more tobacco plants genotyped in step (b) that contain the enhanced nitrogen use efficiency allele.

[0432] Embodiment 163. The method of embodiment 161 or 162, further comprising:

[0433] d. hybridizing one or more tobacco plants selected in step (c) with a second tobacco plant; and

[0434] e. Obtaining progeny seeds from the cross of step (d).

[0435] Embodiment 164. The method of any one of embodiments 161-163, wherein the first population of tobacco plants is of the Maryland variety.

[0436] Embodiment 165. The method of any one of embodiments 161-164, wherein the first tobacco plant population is a variety selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925.

[0437] Embodiment 166. The method of any one of embodiments 161-165, wherein the second tobacco plant is a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0438] Embodiment 167. The method of any one of embodiments 161-166, wherein the progeny seeds comprise the molecular marker.

[0439] Embodiment 168. The method of any one of embodiments 161-167, wherein the progeny seeds comprise the enhanced nitrogen use efficiency.

[0440] Embodiment 169. A method of introgressing an enhanced nitrogen use efficiency trait into a tobacco variety, comprising:

[0441] a. hybridizing a first tobacco variety comprising the enhanced nitrogen use efficiency trait and a second tobacco variety lacking the enhanced nitrogen use efficiency trait;

[0442] b. Progeny seeds obtained from the hybridization of step (a);

[0443] c. genotyping at least one of the progeny seeds obtained in step (b) for a molecular marker associated with the enhanced nitrogen use efficiency trait, wherein the molecular marker is within 20 cM of a locus selected from the group consisting of SEQ ID NOs: 9-16; and

[0444] d. Selecting progeny seeds comprising the enhanced nitrogen use efficiency trait.

[0445] Embodiment 170. The method of embodiment 169, wherein the first tobacco variety is a Maryland tobacco variety.

[0446] Embodiment 171. A method according to embodiment 169 or 170, wherein the first tobacco variety is a variety selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, NC925.

[0447] Embodiment 172. The method of any one of embodiments 169-171, wherein the second tobacco variety is a Burley variety.

[0448] Embodiment 173. The method of any one of embodiments 169-172, wherein the second tobacco variety is a variety selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, TN97LC.

[0449] Embodiment 174. The method of any one of claims 169-173, wherein the enhanced NUE comprises increased partial factor productivity (PFP) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0450] Embodiment 175. The method of any one of embodiments 169-174, wherein the enhanced NUE comprises increased agronomic efficiency (AE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0451] Embodiment 176. The method of any one of embodiments 169-175, wherein the enhanced NUE comprises increased recovery efficiency (RE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0452] Embodiment 177. The method of any one of embodiments 169-176, wherein the enhanced NUE comprises increased physiological efficiency (PE) compared to a tobacco plant lacking the enhanced NUE grown under identical conditions.

[0453] Embodiment 178. The method of any one of embodiments 169-177, wherein the enhanced NUE comprises increased internal efficiency (IE) compared to a tobacco plant lacking the enhanced NUE grown under the same conditions.

[0454] Embodiment 179. A method according to any one of embodiments 169-178, wherein the molecular marker is within 15 cM of the locus.

[0455] Embodiment 180. A method according to any one of embodiments 169-179, wherein the molecular marker is within 10 cM of the locus.

[0456] Embodiment 181. A method according to any one of embodiments 169-180, wherein the molecular marker is within 5 cM of the locus.

[0457] Embodiment 182. A method according to any one of embodiments 169-181, wherein the molecular marker is within 2 cM of the locus.

[0458] Embodiment 183. A method according to any one of embodiments 169-182, wherein the molecular marker is within 1 cM of the locus.

[0459] Embodiment 184. A method according to any one of embodiments 169-183, wherein the molecular marker is within 0.5 cM of the locus.

[0460] Embodiment 185. A method of selecting a tobacco plant comprising an enhanced nitrogen use efficiency trait, comprising:

[0461] a. Isolation of nucleic acids from tobacco germplasm collections;

[0462] b. Determining the nucleic acid for one or more markers located within 20 cM of a locus selected from the group consisting of: SEQ ID NOs: 9-16; and

[0463] c. selecting said tobacco plants comprising said enhanced nitrogen use efficiency trait.

[0464] Embodiment 186. The method of embodiment 185, further comprising:

[0465] d. hybridizing the tobacco plant selected in step (c) with a second tobacco plant; and

[0466] e. Obtaining progeny seeds from the cross of step (d).

[0467] Embodiment 187. A method according to embodiment 185 or 186, wherein the molecular marker is within 15 cM of the locus.

[0468] Embodiment 188. A method according to any one of embodiments 185-187, wherein the molecular marker is within 10 cM of the locus.

[0469] Embodiment 189. A method according to any one of embodiments 185-188, wherein the molecular marker is within 5 cM of the locus.

[0470] Embodiment 190. A method according to any one of embodiments 185-189, wherein the molecular marker is within 2 cM of the locus.

[0471] Embodiment 191. A method according to any one of embodiments 185-190, wherein the molecular marker is within 1 cM of the locus.

[0472] Embodiment 192. A method according to any one of embodiments 185-191, wherein the molecular marker is within 0.5 cM of the locus.

[0473] Embodiment 193. A method of selecting a tobacco plant comprising an enhanced nitrogen use efficiency trait, comprising:

[0474] a. Isolation of nucleic acids from tobacco germplasm collections;

[0475] b. Determining the nucleic acid for one or more markers located within 20 cM of a SNP marker selected from the group consisting of: SEQ ID NOs: 57-64; and

[0476] c. selecting said tobacco plants comprising said enhanced nitrogen use efficiency trait.

[0477] Embodiment 194. A method according to embodiment 193, wherein the determination comprises determining the G nucleotide at position 57 of SEQ ID NO:58.

[0478] Embodiment 195. A method according to embodiment 193 or 194, wherein the determination comprises determining the C nucleotide at position 117 of SEQ ID NO:58.

[0479] Embodiment 196. A method according to any one of embodiments 193-195, wherein the determination comprises determining the G nucleotide at position 57 and the C nucleotide at position 117 of SEQ ID NO:58.

[0480] Embodiment 197. A method according to any one of embodiments 193-195, wherein the determination comprises determining the T nucleotide at position 14 of SEQ ID NO:57.

[0481] Embodiment 198. A method according to any one of embodiments 193-197, wherein the determination comprises determining the G nucleotide at position 162 of SEQ ID NO:59.

[0482] Embodiment 199. A method according to any one of embodiments 193-198, wherein the determination comprises determining the C nucleotide at position 36 of SEQ ID NO:60.

[0483] Embodiment 200. A method according to any one of embodiments 193-199, wherein the determining comprises determining the T nucleotide at position 36 of SEQ ID NO:61.

[0484] Embodiment 201. A method according to any one of embodiments 193-200, wherein the determination comprises determining the T nucleotide at position 36 of SEQ ID NO:62.

[0485] Embodiment 202. A method according to any one of embodiments 193-201, wherein the determination comprises determining the G nucleotide at position 36 of SEQ ID NO:63.

[0486] Embodiment 203. A method according to any one of embodiments 193-202, wherein the determination comprises determining the T nucleotide at position 36 of SEQ ID NO:64. DETAILED DESCRIPTION

[0487] Example 1: Field production practice

[0488] Field-grown tobacco plants were produced using standard field production practices. Each test plot contained up to 40 rows of transplanted seedlings. Prior to transplanting, the seedlings were germinated in a greenhouse. To test the NUE trait, the test plots received a nitrogen rate of 60 pounds of nitrogen per acre. When 50% of the plants in the test plots reached the elongated button stage, the plants were topped using standard procedures. Pesticide application followed a standard protocol. Leaves were harvested at maturity and divided into three branches per plot, with five plants per branch for ripening. Leaves were sampled from the branches at the removal / stripping stage. Five leaves were harvested from three different branches for each experimental variety, with 15 leaves per sample. Half of the leaves were harvested from the fourth leaf at the top of each plant for sampling. Analyses for alkaloids, TSNAs, and NO3 were performed using conventional methods known in the art.

[0489] Example 2: Identification of metabolites associated with enhanced nitrogen utilization

[0490] The Maryland tobacco variety requires approximately 25% less nitrogen fertilizer input compared to the Burley tobacco variety.To identify metabolites associated with high nitrogen efficiency (Maryland) and low nitrogen efficiency (Burley) tobacco varieties, differences in metabolite levels were examined in the Maryland tobacco variety MD609 and the Burley tobacco variety TN90.

[0491] MD609 and TN90 seedlings were germinated from seeds and grown for six weeks without nitrogen supplementation. After six weeks, seedlings of each variety were divided into two groups: Group A contained plants supplied with 0.01% nitrogen or normal greenhouse fertilizer, and Group B contained plants supplied with 25 ppm nitrogen or 25% of the normal greenhouse fertilizer rate. Metabolites were extracted from root and leaf tissues using methanol at 10 and 14 weeks after sowing.

[0492] The isolated metabolites were analyzed using three different LC / MS approaches (UHPLC-MS / MS (+ESI), UHPLC-MS / MS (-ESI), and GC-MS (+EI)) to isolate and identify individual metabolites. Metabolites were identified by comparing the obtained mass spectra with a standard spectral database (Metabolon Inc, Morrisville, NC). Peaks were quantified using the area under the curve. Each compound was calibrated by recording the median as equal to one (1.00) and normalizing each data point proportionally (called "block correction"). The molecular weights of the unknown metabolites are provided in Table 1. Tables 2-5 list the distinguishing metabolites, along with the calibrated measurements for each sample. Discriminating metabolites were determined by comparing the Student's t-test between TN90 and MD609 considering all time points. Metabolites with a p-value less than 0.01 were included in the analysis.

[0493] Table 1: Molecular masses of unknown metabolite compounds expressed in kilodaltons

[0494] metabolites quality X-21756 247.0918 X-21796 138.0566 X-23319 299.0771 X-23330 251.1136 X-23366 189.1023 X-23389 157.0762 X-23453 161.0818 X-23454 319.0933 X-23576 267.1237 X-23580 311.1136 X-23852 374.144 X-23916 395.0291 X-23937 161.0819

[0495] Table 2: Metabolites negatively correlated with enhanced nitrogen efficiency identified in root tissue when comparing MD609 and TN90 tobacco lines at 10 and 14 weeks after sowing.

[0496]

[0497] Table 3: Metabolites positively correlated with enhanced nitrogen efficiency identified in root tissue when comparing MD609 and TN90 tobacco lines

[0498]

[0499] Table 4: Metabolites negatively correlated with enhanced nitrogen efficiency identified in leaf tissue when comparing MD609 and TN90 tobacco lines.

[0500]

[0501] Table 5: Metabolites positively correlated with enhanced nitrogen efficiency identified in leaf tissue when comparing MD609 and TN90 tobacco lines

[0502]

[0503] Example 3: Identification of gene expression associated with enhanced nitrogen use efficiency

[0504] RNA extraction was also performed on the same plants used in Example 1 for RNAseq. RNA was extracted from leaf and root tissues at 10 and 14 weeks after sowing for Illumina sequencing. RNAseq data were analyzed according to standard methods in the art. Subsequently, candidate genes were validated.

[0505] Seventeen genes (tables 6 and 7) were found to be negatively correlated with the enhanced nitrogen utilization efficiency phenotype of MD609, and seven genes (tables 8 and 9) were found to be positively correlated with the enhanced nitrogen utilization efficiency phenotype of MD609. The negatively correlated genes were candidate genes (through mutagenesis, cisgene conversion or transgenic transformation) that were down-regulated in the burley tobacco variety, and the positively correlated genes were candidate genes that were overexpressed to improve nitrogen utilization efficiency in the burley tobacco variety. Relevant single nucleotide polymorphism (SNP) markers were provided to track each candidate gene (tables 6-10). The polymorphisms associated with the MD609 allele were provided, which were therefore conducive to enhanced NUE (table 10).

[0506] Identification of the genomic location of each associated gene identified four gene clusters associated with enhanced NUE in the tobacco genome ( Figure 1 Seven genes were similarly located on chromosome 1, four genes were similarly located on chromosome 11, three genes were similarly located on chromosome 14, and five genes were similarly located on chromosome 20 ( Figure 1 These four positions are also hotspots of differentially expressed genes under low nitrogen and normal nitrogen conditions ( Figure 1 ). SNP markers were generated to identify MD609 as being specific, and thus enhanced NUE polymorphisms were identified for each of these positions (Tables 6-10). Further characterization of the region on chromosome 11 revealed that it contained 79 total expressed genes, and 46 of these genes were differentially expressed under low nitrogen conditions ( Figure 2 ).

[0507] Table 6: Genes identified as negatively correlated with enhanced nitrogen use efficiency in root tissue.

[0508]

[0509] Table 7: Genes identified as negatively correlated with enhanced nitrogen use efficiency in leaf tissue.

[0510]

[0511] Table 8: Genes identified as positively correlated with enhanced nitrogen use efficiency in root tissue.

[0512]

[0513] Table 9: Genes identified as positively correlated with enhanced nitrogen use efficiency in leaf tissue.

[0514]

[0515] Table 10: SNP markers containing polymorphisms associated with enhanced NUE

[0516]

[0517]

[0518] Example 4: Identification of tobacco leaf-preferred and root-preferred promoters

[0519] RNA samples from 4-week-old TN90 tobacco plants were obtained from 10 tissue types (axillary buds before topping; axillary buds 2 hours after topping; axillary buds 6 hours after topping; axillary buds 24 hours after topping; axillary buds 72 hours after topping; roots before topping; roots 24 hours after topping; roots 72 hours after topping; young leaves during topping; and shoot apical meristem). The resulting RNA samples (three independently collected samples for each tissue type) were used as starting material for Illumina 1x100bp sequencing.

[0520] Illumina reads were mapped and used to identify candidate gene lists that exhibited high root or leaf expression. Tables 11 and 12 provide RPKM expression values ​​for genes identified as having leaf-preferred or root-preferred expression. These genes are candidate genes with leaf-preferred or root-preferred promoters, respectively.

[0521] Table 11: Genes with leaf-preferred expression

[0522]

[0523] Table 12: Genes with root-preferred expression

[0524]

[0525] Example 5: Development of modified plants

[0526] The expression vector p45-2-7 (SEQ ID NO: 65) was used as a backbone to generate multiple transformation vectors (see Examples XY). p45-2-7 contains the CsVMV promoter, the NOS terminator, and a cassette containing the kanamycin selectable marker (NPT II) operably linked to the Actin2 promoter and NOS terminator. The nucleic acid vector containing the desired transgene was introduced into tobacco leaf discs via Agrobacterium transformation. For example, see Mayo et al., 2006, Nat Protoc. 1: 1105-11 and Horsch et al., 1985, Science 227: 1229-1231.

[0527] In Magenta TM Grow TN90 tobacco plants in the GA-7 box, cut leaf disc and be placed in culture plate.By in 50mL centrifuge tube, collect the Agrobacterium tumefaciens (Agrobacterium tumefaciens) cell that comprises transformation carrier with 3500RPM centrifugal 20mL cell suspension 10 minutes.Remove supernatant, Agrobacterium tumefaciens cell precipitation is resuspended in 40mL liquid resuspension culture medium.With #15 razor blade, tobacco leaf is cut into 8 0.6cm disks (avoiding midrib), and is inverted in culture plate.The thin layer Murashige & Skoog with B5 vitamin liquid resuspension culture medium is added in culture plate, and pierce leaf disc evenly with fine pin.About 25mL Agrobacterium tumefaciens suspension is added in culture plate, and leaf disc is hatched 10 minutes in suspension.

[0528] The leaf disc is transferred to a co-cultivated culture plate (1 / 2MS culture medium), the disc is placed upside down, and contacted with a filter paper covered on a co-cultivated TOM culture medium (containing 20g / L sucrose; 1mg / L indole-3-acetic acid and 2.5mg / L 6-benzylaminopurine (BAP) MS culture medium). The culture plate is sealed with a sealing film, then incubated at 24°C for 6 hours in dim light (60-80mE / ms) with a photoperiod of 18 hours of light and 6 hours of darkness for 3 days. After incubation, the leaf disc is transferred to a culture plate (TOM culture medium plus 300mg / L kanamycin) of regeneration / selection TOM K culture medium. The leaf disc is passaged to fresh TOM K culture medium every two weeks at 24°C in dim light with a photoperiod of 18 hours of light and 6 hours of darkness until the bud becomes excised. Remove the bud from the leaf with tweezers and insert it into the MS basal medium containing 100mg / L kanamycin. Shoots were incubated on MS basal medium containing 100 mg / L kanamycin at 24°C with a photoperiod of 18 h light and 6 h dark (high intensity light, 6080 mE / ms) to induce rooting.

[0529] When the plantlets containing shoots and roots are large enough (e.g., reaching Magenta TM The nitrogen use efficiency of the modified plants (T0, T1, T2 or later generations) and control plants were evaluated. The control plants were untransformed NLM plants or NLM plants transformed with empty p45-2-7 vectors.

[0530] Phenotypic screening for enhanced nitrogen use efficiency was performed in the greenhouse using zero parts per million (ppm) nitrogen (no nitrogen), 25 ppm nitrogen (low nitrogen), and 100 ppm nitrogen (normal nitrogen). The initial screening was performed in a greenhouse with T1 plants. Then, the homozygous T2 population was assessed in the field using 60 lbs / acre fertilizer (approximately 25% of the recommended rate for burley tobacco). Seedling growth, chlorophyll loss, and final yield were measured and compared to control plants grown under normal nitrogen levels.

[0531] In the T1 generation, plants overexpressing G20580 (2 independent transformants), G42290 (4 independent transformants), G41446 (4 independent transformants), G53261 (2 independent transformants), and G30999 (3 independent transformants) were grown in the greenhouse, along with controls under nitrogen limiting conditions equivalent to 60 lbs nitrogen / acre. Nine plants were sampled for each transformant, and one of the lines overexpressing G41446 showed a statistically significant increase in yield (fresh weight grams / plant) compared to the control (see Figure 5 ).

[0532] Example 6: Production of Cisgenic Tobacco Plants with Enhanced Nitrogen Utilization Efficiency

[0533] Nitrogen use efficiency can be improved by modifying gene expression of genes identified as differentially expressed in Example 2. Similarly, genes involved in the biosynthesis or degradation of metabolites identified in Example 1 can be regulated to improve nitrogen use efficiency. Genes positively correlated with enhanced nitrogen use efficiency can be overexpressed using general overexpression promoters or tissue-preferred promoters to overexpress the gene in the desired tissue.

[0534] Generate transformation vectors to overexpress proteins that are positively correlated with enhanced nitrogen utilization efficiency. A separate transformation vector comprising one of SEQ ID NOs: 9-16 is incorporated into the p45-2-7 transformation vector. Additionally, a transformation vector comprising one of SEQ ID NOs: 9-16 is generated.

[0535] These transformation vectors were used to generate modified tobacco plants according to Example 4. The modified tobacco plants (T1 generation) and control tobacco plants were then phenotypically evaluated as described in Example 4. The modified tobacco plants exhibited enhanced nitrogen use efficiency compared to control tobacco plants grown under the same conditions.

[0536] Example 7: Generation of transgenic tobacco plants with enhanced nitrogen use efficiency

[0537] Nitrogen utilization efficiency can also be enhanced by down-regulating the expression of genes identified in Example 2 as negatively correlated with nitrogen utilization efficiency.

[0538] Transformation vectors containing RNAi constructs were designed to suppress the tobacco genes whose expression in Example 2 was negatively correlated with nitrogen use efficiency. A separate transformation vector comprised one of SEQ ID NOs:41-56, which was incorporated into the p45-2-7 transformation vector. Additional transformation vectors were generated, which comprised one of SEQ ID NOs:41-56.

[0539] These transformation vectors were used to generate modified tobacco plants according to Example 4. The modified tobacco plants (T1 generation) and control tobacco plants were then phenotypically evaluated as described in Example 4. The modified tobacco plants exhibited enhanced nitrogen use efficiency compared to control tobacco plants grown under the same conditions.

[0540] Example 8: Other methods for improving nitrogen use efficiency using gene editing technology

[0541] Gene editing technologies such as CRISPR / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, CRISPR / Csm1, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used to modify the gene coding region that is negatively correlated with enhanced nitrogen utilization efficiency so that the gene encodes a non-functional protein or a low-functional protein. These gene editing technologies are also used to edit or replace endogenous promoter sequences to drive their homologous protein expression in leaf or root tissues, thereby improving nitrogen utilization efficiency. For example, endogenous G64360 is edited or replaced so that the gene is only expressed in leaf tissue, where it can work to improve the nitrogen utilization efficiency of plants.

[0542] Separate CRISPR / Cas9 or CRISPR / Cpf1 guide RNAs were constructed to recognize and hybridize with the promoter sequences of each of SEQ ID NOs: 9-40. The engineered guide RNAs and a donor polynucleotide comprising a promoter selected from the group consisting of SEQ ID NOs: 17-24 were provided to tobacco plants, allowing the selected promoter to replace the endogenous promoter of the selected gene and restrict endogenous expression to leaf or root tissue as desired. The edited tobacco plants exhibited enhanced nitrogen use efficiency compared to control tobacco plants grown under similar conditions.

[0543] Example 9: Development of new mutations via random mutagenesis to improve nitrogen use efficiency

[0544] Random mutagenesis of tobacco plants was performed using either ethyl methanesulfonate (EMS) mutagenesis or fast neutron bombardment. EMS mutagenesis involves chemically inducing random point mutations. Fast neutron mutagenesis involves exposing seeds to neutron bombardment, which causes large deletions through double-strand DNA breaks.

[0545] For EMS mutagenesis, 1 gram (approximately 10,000 seeds) burley tobacco (kind TN90) seed was washed 15 minutes in 0.1% Tween, then soaked 2 hours in 30mL ddH o.Then, 150 μ L of 0.5% EMS (Sigma, catalog number (Cat. No.) M-0880) was mixed into seed / ddH in the O solution and at room temperature (RT; Approximately 20 ℃) ​​under the hood, hatched 8-12 hour (rotating with 30RPM).Then, remove the liquid in the seed, and mix it into the 1M NaOH and spend the night to purify and remove.Then, use 100mL ddH o washed seeds twice, continued 2-4 hour.Then, washed seeds were suspended in 0.1% agar solution.

[0546] EMS-treated seeds in an agar solution were evenly spread at ~2,000 seeds / plate on Carolina's Choice Tobacco Mix (Carolina Soil Company, Kinston, NC) in water-soaked plates. The plates were then covered with plastic wrap and placed in a growth chamber. Once the seedlings emerged from the soil, the plastic wrap was punctured to allow the humidity to gradually decrease. After two weeks, the plastic wrap was completely removed. The plates were moved to a greenhouse and fertilized with NPK fertilizer. The seedlings were reinserted into floating trays and grown until they were transplant size. The plants were then transplanted into the field. During the growth period, the plants self-pollinated to form M1 seeds. At maturity, five pods were harvested from each plant, and a group of seeds from each plant was individually named. This formed the M1 population. A composite of M1 seeds from each M0 plant was grown, and the plants were phenotypically evaluated for enhanced nitrogen efficiency as described in Example 4. M1 plants exhibiting enhanced nitrogen efficiency were selected, and mutations were screened using DNA sequencing and gene mapping techniques known in the art.

[0547] Example 10: Using breeding to produce tobacco plants with enhanced nitrogen use efficiency

[0548] Conventional breeding techniques can be used to introduce the favorable NUE alleles provided herein into any tobacco variety to enhance NUE. A tobacco plant population can be generated by hybridizing tobacco plants having at least one favorable NUE allele (see Table 10) with tobacco plants lacking the favorable allele. Marker-assisted selection or other techniques known in the art (e.g., direct sequencing) can be used to track the introgression of favorable alleles in the F1 generation and can be used to determine heterozygosity or homozygosity in subsequent generations. Enhanced NUE in offspring plants can be determined using methods known in the art or described above. Multiple different favorable NUE alleles can be combined into a single strain. Molecular phenotypes determined by metabolite profiles can be used to track enhanced NUE during breeding. Metabolite profiles of offspring plants can be determined using the methods described above. Offspring plants with metabolic profiles of parent plants with enhanced NUE are hybridized to produce subsequent populations of tobacco plants with enhanced NUE.

[0549] Introduction of the Maryland609 locus into commercially available Burley tobacco varieties to develop Burley tobacco lines with enhanced NUE can be performed as described. Screening of 23 Burley tobacco and 6 MD609 lines identified 3 Burley tobacco lines containing the MD609 allele at SNP marker S451 (SEQ ID NO: 58) ( Figure 3Three burley tobacco lines carrying the MD609 allele were tested for chlorophyll loss, growth, and yield under nitrogen-limiting conditions and compared with a control TN90 burley tobacco line and a control MD609 line (MD609 carrying the MD609 allele at SNP marker S451). Figure 4 Burley lines carrying the MD609 allele appeared more similar to the Maryland control ( Figure 4 ) chlorophyll loss, growth and yield. The TN90 burley control showed increased chlorophyll loss, decreased growth and reduced yield compared to the MD609 control ( Figure 4 These results indicate that the introduction of the MD609 allele at SNP marker S451 can enhance NUE.

[0550] To introduce the MD609 allele into Burley tobacco, MD609 was crossed with Burley tobacco. The F1 progeny from this cross were selected and subsequently selfed to produce F2 seed. The F2 and F3 plants were grown and selfed to produce F4 seed. Swollen F4 seeds from two independent crossing schemes identified as NUE-2 and NUE-3 lines, respectively, were grown and harvested in the field. For the F4 seeds of the NUE-2 and NUE-3 lines, the genotypes for the SNP markers S451, S317, S12385, S238, S3894, and S2237 were determined (see Table 13). The F4 plants were grown using the reduced nitrogen production method described in Example 1. Both the NUE-2 and NUE-3 lines showed increased yield in pounds per acre compared to the Burley tobacco control TN90 (see Table 13). Figure 6 ).

[0551] Alternatively, modified tobacco plants comprising an enhanced NUE phenotype can be generated using the methods described herein and hybridized with unmodified tobacco plants to propagate the modification in subsequent generations. Selection of genetic modifications can be tracked using appropriate techniques known in the art. Enhanced NUE in progeny plants can be determined using methods known in the art or described above.

[0552] Table 13: Genotypes of field grown plants from F4 NUE-2 and NUE-3 lines and TN90. MD stands for MD609 allele, Burley stands for Burley allele, and HET stands for heterozygous MD609 / Burley.

[0553] S451 S317 S12835 S238 S3894 S2237 NUE-2 MD HET MD Burley tobacco Burley tobacco MD NUE-3 MD HET MD Burley tobacco Burley tobacco MD TN90 Burley tobacco Burley tobacco Burley tobacco Burley tobacco Burley tobacco Burley tobacco

Claims

1. A method of producing a tobacco plant comprising an enhanced nitrogen use efficiency (NUE) trait, the method comprising: a. Providing a first tobacco plant population comprising an enhanced NUE trait; b. Genotyping the first tobacco plant population for the presence of one or more molecular markers located within 5 cM of a SNP marker associated with an enhanced NUE trait, the SNP marker comprising SEQ ID NO: 58 and having polymorphic position 57 allele G and polymorphic position 117 allele C; c. selecting tobacco plants comprising said one or more molecular markers; d. hybridizing the tobacco plant selected in step (c) with a second tobacco plant; and e. Obtaining progeny seeds from the cross of step (d), wherein a plant grown from the progeny seeds comprises the enhanced NUE trait and the SNP marker.

2. The method of claim 1 , wherein the enhanced NUE trait is selected from the group consisting of increased partial factor productivity (PFP), increased agronomic efficiency (AE), increased recovery efficiency (RE), increased physiological efficiency (PE), and increased internal efficiency (IE), compared to a tobacco plant lacking the enhanced NUE trait grown under the same conditions.

3. The method of claim 1, wherein the first tobacco plant population is selected from the group consisting of MD609, MD601, Banket A1, K326, K346, K358, K394, K399, K730, NC196, NC37NF, NC471, NC55, NC92, NC2326, NC95, and NC925.

4. The method of claim 1, wherein the second tobacco plant is a Burley tobacco variety.

5. The method of claim 1, wherein the second tobacco plant is selected from the group consisting of TN86, TN86LC, TN90, TN90LC, TN97, and TN97LC. The method of claim 1 , wherein the one or more molecular markers are within 3 cM of the SNP marker. The method of claim 1 , wherein the one or more molecular markers are within 1 cM of the SNP marker.

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