Compositions and methods based on PMT operations for producing tobacco plants and products with altered alkaloid levels

Abstract

The present invention provides compositions and methods relating to tobacco plants having altered total alkaloid and nicotine levels and commercially acceptable leaf grades, their development through breeding or transgenic approaches, and the production of tobacco products from such tobacco plants. [Solution] A tobacco plant or a part thereof containing one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant, when grown and processed under equivalent conditions, can produce leaves with lower total alkaloid levels and nicotine levels compared to a control tobacco plant that does not have the one or more mutant alleles, is provided as a tobacco plant or a part thereof, and a dried tobacco product derived from the tobacco plant.

Description

Technical Field

[0001] Incorporation of Sequence Listing This application claims the benefit of U.S. Provisional Application No. 62 / 703,775, filed Jul. 26, 2018, and U.S. Provisional Application No. 62 / 848,159, filed May 15, 2019, both of which are hereby incorporated by reference in their entirety. The sequence listing contained in the file named "P34620WO00_SL.txt", which is 200,338 bytes (measured in MS-Windows®), was created on Jul. 25, 2019, and is hereby submitted electronically as an appendix hereto and incorporated by reference in its entirety.

[0002] Field The present disclosure provides for genetic engineering of tobacco to regulate alkaloid and nicotine levels.

Background Art

[0003] Background Nicotine is the main alkaloid and typically accounts for over 90-95% of the total alkaloids in commercially available tobacco cultivars. The remaining alkaloid fraction consists mainly of three additional alkaloids: nornicotine, anabasine, and anatabine. By modulating different nicotine biosynthesis genes and transcriptional regulators, tobacco plants with reduced nicotine levels have been obtained, but the results have been variable and inconsistent. There is a need for new technologies to reduce the nicotine level in tobacco leaves.

Summary of the Invention

[0004] Summary The present disclosure provides tobacco plants having altered total alkaloid and nicotine levels and commercially acceptable leaf grades, their development via breeding or transgenic approaches, and the manufacture of tobacco products from such tobacco plants.

[0005] In one embodiment, the disclosure provides a tobacco plant or a portion thereof containing one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, the tobacco plant being able to produce leaves containing a lower nicotine level than that of leaves from a control tobacco plant that does not have one or more mutant alleles, when grown and processed under equivalent conditions.

[0006] In another embodiment, the tobacco plant contains one or more mutant alleles of at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

[0007] In a further embodiment, the tobacco plant contains one or more mutant alleles of at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

[0008] In another embodiment, the tobacco plant contains one or more mutant alleles of at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

[0009] In a further embodiment, the tobacco plant contains one or more mutant alleles of five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

[0010] In one embodiment, the present disclosure provides tobacco plants selected from the group consisting of single pmt mutants, double pmt mutants, triple mutants, quadruple mutants, and quintuple mutants, as listed in Tables 8A to 8E.

[0011] In one embodiment, the disclosure provides tobacco plants listed in Tables 4A to 4E or Table 10. In another embodiment, the disclosure provides offspring plants of tobacco plants in Tables 4A to 4E or Table 10, derived either from self-pollination or hybridization with another plant.

[0012] In another embodiment, the disclosure provides tobacco plants comprising various combinations of pmt mutant alleles listed in Tables 5A-5E or Tables 12A-12E that result in single pmt mutants, double pmt mutants, triple mutants, quadruple mutants, or quintuple mutants. In one embodiment, the disclosure provides tobacco plants comprising sequences of pmt mutant alleles selected from the group consisting of SEQ ID NOs: 21-200, 410-441, 474-505, 538-569, 602-633, and 666-697.

[0013] This disclosure further provides tobacco products comprising cured tobacco, tobacco blends, and plant materials derived from disclosed tobacco plants, strains, varieties, or hybrids. [Invention 1001] A tobacco plant or a part thereof comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant, when grown and processed under equivalent conditions, can produce leaves containing a lower nicotine level than that of leaves from a control tobacco plant that does not have the one or more mutant alleles. [Invention 1002] The tobacco plant of the present invention 1001 or a part thereof, comprising one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [Invention 1003] The tobacco plant of the present invention 1001 or a part thereof, comprising one or more mutant alleles of at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [Invention 1004] The tobacco plant of the present invention 1001 or a part thereof, comprising one or more mutant alleles of at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [Invention 1005] The tobacco plant of the present invention 1001 or a part thereof, comprising one or more mutant alleles of five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [Invention 1006] The tobacco plant or a part thereof according to any of the inventions 1001 to 1005, which, when grown and processed under equivalent conditions, can produce leaves containing nicotine levels lower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the nicotine levels of the leaves of a control tobacco plant that does not have the one or more mutant alleles. [Invention 1007] The tobacco plant or any part thereof according to any of the inventions 1001 to 1006, which, when grown and processed under equivalent conditions, can produce leaves containing a total alkaloid level lower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the total alkaloid level of the leaves from the control tobacco plant. [Invention 1008] The tobacco plant or a part thereof according to Invention 1007, wherein the tobacco plant, when grown and processed under equivalent conditions, can produce leaves containing a total alkaloid level lower than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid level of the leaves from the control tobacco plant. [Invention 1009] A tobacco plant or part thereof according to any of the inventions 1001 to 1008, wherein the one or more mutant alleles include a mutation in a sequence region selected from the group consisting of the promoter, 5'UTR, first exon, first intron, second exon, second intron, third exon, 3'UTR, terminator, and any combination thereof. [Invention 1010] A tobacco plant or part thereof according to any of the inventions 1001 to 1009, wherein the one or more mutant alleles include one or more mutant types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof. [Invention 1011] The one or more mutant alleles mentioned above are as follows: PMT protein cleavage, non-translating PMT gene transcripts, non-functional PMT proteins, immature stop codons in the PMT gene, and any combination thereof. A tobacco plant or part thereof according to any of the inventions 1001 to 1010, which gives rise to one or more of the following: [Invention 1012] A tobacco plant or part thereof according to any of the invention 1001 to 1011, wherein the one or more mutant alleles contain a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the wild-type PMT gene. [Invention 1013] A tobacco plant or a part thereof according to any of the invention 1001 to 1012, wherein the one or more mutant alleles include a conjugation state selected from the group consisting of homozygosity, heterozygosity, and heteroallelemicity. [Invention 1014] A tobacco plant or a part thereof according to any of the invention 1001 to 1012, wherein one or more mutant alleles are homozygous or heteroallelic in at least 1 to 5 PMT genes. [Invention 1015] The tobacco plant or a part thereof according to any one of Inventions 1001 to 1012, wherein the one or more mutant alleles are homozygous or heterozygous in at least four PMT genes. [Invention 1016] The tobacco plant or a part thereof according to any one of Inventions 1001 to 1012, wherein the one or more mutant alleles are homozygous or heterozygous in all five PMT genes. [Invention 1017] The tobacco plant or a part thereof according to any one of Inventions 1001 to 1016, wherein the at least two PMT genes are PMT1a and PMT3. [Invention 1018] The tobacco plant or a part thereof according to any one of the above Inventions, which can produce leaves containing a nicotine level selected from the group consisting of less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01% by dry weight. [Invention 1019] The tobacco plant or a part thereof according to any one of the above Inventions, which can produce leaves containing a total alkaloid level selected from the group consisting of less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2% by dry weight. [Invention 1020] The tobacco plant or a part thereof according to any one of the above Inventions, which can produce dry leaves containing a total TSNA level of 2 to 0.05, 1.9 to 0.05, 1.8 to 0.05, 1.7 to 0.05, 1.6 to 0.05, 1.5 to 0.05, 1.4 to 0.05, 1.3 to 0.05, 1.2 to 0.05, 1.1 to 0.05, 1.0 to 0.05, 0.9 to 0.05, 0.8 to 0.05, 0.7 to 0.05, 0.6 to 0.05, 0.5 to 0.05, 0.4 to 0.05, 0.3 to 0.05, 0.2 to 0.05, 0.15 to 0.05, or 0.1 to 0.05 ppm. [Invention 1021] A population of tobacco plants of any one of the inventions 1001 to 1020. [Invention 1022] A dried tobacco material derived from a tobacco plant of any one of the inventions 1001 to 1020. [Invention 1023] The dried tobacco material of Invention 1022 produced by a drying process selected from the group consisting of hot air drying, air drying, direct fire drying, and sun drying. [Invention 1024] Containing tobacco leaves, The tobacco leaves showing reduced mold infection as compared to a control dried tobacco material derived from variety LA Burley 21, The dried tobacco material of Invention 1022. [Invention 1025] A tobacco blend containing the dried tobacco material of Invention 1022. [Invention 1026] The dried tobacco material constitutes at least about 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, or at least 95 wt% of the dried tobacco in the tobacco blend. The tobacco blend of Invention 1025. [Invention 1027] The dried tobacco material constitutes at least about 10 vol%, at least 15 vol%, at least 20 vol%, at least 25 vol%, at least 30 vol%, at least 35 vol%, at least 40 vol%, at least 45 vol%, at least 50 vol%, at least 55 vol%, at least 60 vol%, at least 65 vol%, at least 70 vol%, at least 75 vol%, at least 80 vol%, at least 85 vol%, at least 90 vol%, or at least 95 vol% of the dried tobacco in the tobacco blend. The tobacco blend of Invention 1025. [Invention 1028] A tobacco product comprising the dried tobacco material of the present invention 1022. [Invention 1029] Tobacco products of the present invention 1028, selected from the group consisting of cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. [Invention 1030] A tobacco product according to the present invention 1028, which is a smokeless tobacco product. [Invention 1031] The smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff, and is a tobacco product of the present invention 1030. [Invention 1032] Reconstituted tobacco comprising the dried tobacco material of the present invention 1022.

[0014] A brief explanation of arrays Sequence IDs 1-5 describe exemplary genomic sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4 from the TN90 reference genome, respectively.

[0015] Sequence IDs 6-10 describe exemplary cDNA sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4 from TN90, respectively.

[0016] Sequence IDs 11-15 describe exemplary polypeptide sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4 from TN90, respectively.

[0017] Sequence IDs 16-22 show exemplary guide RNA sequences.

[0018] Sequence IDs 23-200, 410-441, 474-505, 538-569, 602-633, and 666-697 describe exemplary edited pmt variant sequences. [Brief explanation of the drawing]

[0019] [Figure 1] RNA expression of five PMT genes in TN90 roots. [Figure 2] Nicotine levels in various low-alkaloid strains: CS15 (a quintuple pmt knockout mutant strain CS15 on an NLM (Ph Ph) background, a PMT RNAi transgenic strain on a VA359 background) and a low-nicotine KY171 ("LN KY171") strain (on a KY171 background with nic1 and nic2 double mutations). Each of these was compared to a normal alkaloid control strain: NLM (Ph Ph), VA359, and KY171 background. [Figure 3] Total alkaloid levels in various low-alkaloid strains: CS15, PMT RNAi, and LN KY171. Each of these compared to a normal alkaloid control strain: NLM(Ph Ph), VA359, and KY171 background. [Figure 4] Leaf yield in various low-alkaloid strains: CS15, PMT RNAi, and LN KY171. Each of these was compared to a normal alkaloid control strain: NLM(Ph Ph), VA359, and KY171 background. [Figure 5] Leaf quality in various low-alkaloid strains: CS15, PMT RNAi, and LN KY171. Each of these compared to a normal alkaloid control strain: NLM(Ph Ph), VA359, and KY171 background. [Figure 6A]Photographs showing mold growth on dried tobacco, including TN90LC (Figure 6A), LA BU21 (Figure 6B), TN90 containing an RNAi construct that downregulates PR50 (Figure 6C), TN90 containing an RNAi construct that downregulates PMT genes (Figure 6D), and TN90 containing edits to all five PMT genes (Figure 6E). [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 7] Figures 6A to 6E depict fungal infections observed in the strains examined. [Modes for carrying out the invention]

[0020] Detailed explanation Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Those skilled in the art will recognize many methods that may be used to implement this disclosure. In fact, this disclosure is by no means limited to the methods and materials described. For the purposes of this disclosure, the following terms are defined below:

[0021] For example, any references cited herein, including all patents and publications, are invoked by reference in their entirety.

[0022] As used herein, the singular forms “a,” “an,” and “the” include multiple references unless the context makes it clear. For example, the terms “compound” or “at least one compound” may include multiple compounds, including mixtures thereof.

[0023] When the term "approximately" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the number, such as 10%.

[0024] As used herein, phrases such as “less than,” “more than,” “at least,” “at most,” “approximately,” “below,” “above,” and “about” modify each value in a set when used with a set of numbers. For example, the expression “less than 1%, 2%, or 3%” is equivalent to “less than 1%, less than 2%, or less than 3%.”

[0025] As used herein, the term "tobacco plant" refers to plants of the species Nicotiana tabacum.

[0026] Nicotine biosynthesis in tobacco begins with the methylation of the polyamine putrescine to N-methylputrescine by the enzyme putrescine N-methyltransferase (PMT) using S-adenosylmethionine as a cofactor. This is the step in which the precursor metabolite is involved in nicotine biosynthesis. The PMT enzyme is classified as EC2.1.1.53 under the enzyme classification system. In Nicotiana tabacum, five genes encode putrescine N-methyltransferase, designated PMT1a, PMT1b, PMT2, PMT3, and PMT4. Table 1A lists the genomic DNA sequences, cDNA sequences, and protein sequences of these five PMT genes in the TN90 plant. This disclosure describes compositions and methods used to edit the PMT gene to produce pmt mutant plants having reduced nicotine levels while maintaining leaf quality.

[0027] As used herein, “PMT1b” or “PMT1b gene” refers to a tobacco locus encoding a polypeptide having the exemplary amino acid sequence of TN90 described in Sequence ID No. 11.

[0028] As used herein, “PMT1a” or “PMT1a gene” refers to a tobacco locus encoding a polypeptide having the exemplary amino acid sequence TN90 described in Sequence ID No. 12.

[0029] As used herein, “PMT2” or “PMT2 gene” refers to a tobacco locus encoding a polypeptide having the exemplary amino acid sequence of TN90 described in Sequence ID No. 13.

[0030] As used herein, “PMT3” or “PMT3 gene” refers to a tobacco locus encoding a polypeptide having the exemplary amino acid sequence of TN90 described in Sequence ID No. 14.

[0031] As used herein, “PMT4” or “PMT4 gene” refers to a tobacco locus encoding a polypeptide having the exemplary amino acid sequence of TN90 described in Sequence ID No. 15.

[0032] As used herein, a mutation refers to a genetic modification introduced into a gene to reduce, inhibit, or eliminate the expression or activity of a product encoded by that gene. Such modifications may be in any sequence region of the gene, e.g., the promoter, 5'UTR, exon, intron, 3'UTR, or terminator region. In one embodiment, the mutation is not a natural polymorphism present in a particular tobacco variety or cultivar. As used herein, a "mutant allele" refers to an allele derived from a locus whose allele locus contains the mutation.

[0033] As used herein, “pmt mutant” refers to a tobacco plant containing one or more mutations in one or more PMT genes. A pmt mutant may be a single mutant, a double mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. As used herein, a single, double, triple, quadruple, or quintuple pmt mutant refers to a mutant having modifications in one, two, three, four, or five PMT genes, respectively. A pmt mutant may also be a combination of homozygous, heterozygous, or heteroallelemic mutants in one or more PMT genes.

[0034] Where used herein, gene names or locus names are written in uppercase and italics, for example, PMT1a, PMT1b, PMT2, PMT3, and PMT4. Protein or polypeptide names are written in uppercase and not italicized, for example, PMT1a, PMT1b, PMT2, PMT3, and PMT4. Variant names (to refer to a general variation in a gene or gene group, or to refer to a specific variant allele) are written in lowercase and italics, for example, pmt, pmt1a, pmt1b, pmt2, pmt3, and pmt4.

[0035] In one embodiment, the disclosure provides a tobacco plant or a portion thereof containing one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, the tobacco plant being able to produce leaves containing a lower nicotine level than that of a control tobacco plant without the one or more mutant alleles when grown and processed under comparable conditions. In one embodiment, a single pmt mutant tobacco plant is provided. In another embodiment, a single pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at a level of less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant without the single pmt mutation. In a further embodiment, single-pmt mutant tobacco plants, when grown under similar growth conditions, contain nicotine at levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of control plants without the single-pmt mutant.

[0036] In another embodiment, the tobacco plant contains one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one embodiment, a double pmt mutant tobacco plant is provided. In another embodiment, the double pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant without the double pmt mutation. In a further embodiment, the double PMT mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of a control plant without the double PMT mutation.

[0037] In a further embodiment, the tobacco plant contains one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one embodiment, a triple pmt mutant tobacco plant is provided. In another embodiment, the triple pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant without the triple pmt mutation. In a further embodiment, the triple PMT mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of a control plant without the triple PMT mutation.

[0038] In another embodiment, the tobacco plant contains one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one embodiment, a quadruple pmt mutant tobacco plant is provided. In another embodiment, the quadruple pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant without the quadruple pmt mutation. In a further embodiment, quadruple PMT mutant tobacco plants, when grown under similar growth conditions, contain nicotine at levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of control plants without the quadruple PMT mutation.

[0039] In a further embodiment, the tobacco plant contains one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In one embodiment, a quintuple pmt mutant tobacco plant is provided. In another embodiment, the quintuple pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the nicotine level of a control plant without the quintuple pmt mutation. In a further embodiment, the quintuple PMT mutant tobacco plant, when grown under similar growth conditions, contains nicotine at levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of a control plant without the quintuple PMT mutation.

[0040] In one embodiment, the tobacco plant is a single pmt mutant, double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant, as listed in Tables 8A-8E. In another embodiment, the tobacco plant contains one or more pmt mutant alleles listed in Tables 5A-5E and Tables 12A-12E. Each and all combinations of pmt mutant alleles listed in Tables 5A-5E and Tables 12A-12E are also provided to result in a single pmt mutant, double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant. Each mutant locus may be homozygous or heterozygous, or may contain a combination of heteroalleles. In another embodiment, the tobacco plant contains a combination of pmt mutant genotypes, as shown for each distinct strain listed in Tables 4A-4E and Table 10. In one embodiment, the tobacco plant contains a sequence of a pmt mutant allele selected from the group consisting of SEQ ID NOs: 21-200, 410-441, 474-505, 538-569, 602-633, and 666-697. In another embodiment, the disclosure provides a double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant containing a sequence of a pmt mutant allele selected from the group consisting of SEQ ID NOs: 21-200, 410-441, 474-505, 538-569, 602-633, and 666-697.

[0041] In one embodiment, when grown and processed under equivalent conditions, tobacco plants can produce leaves containing nicotine levels lower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the nicotine levels of control tobacco plants. In another embodiment, when grown and processed under equivalent conditions, tobacco plants can produce leaves containing nicotine levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the nicotine levels of control tobacco plants.

[0042] In another embodiment, when grown and processed under comparable conditions, tobacco plants can produce leaves containing total alkaloid levels lower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the total alkaloid levels of leaves from a control tobacco plant. In another embodiment, when grown and processed under comparable conditions, tobacco plants can produce leaves containing total alkaloid levels of 1%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95% of the total alkaloid levels of a control tobacco plant.

[0043] In a further embodiment, tobacco plants, when grown and processed under comparable conditions, can produce leaves containing total alkaloid levels lower than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid levels of leaves from control tobacco plants.

[0044] In one embodiment, the mutant pmt allele contains a mutation in a PMT sequence region selected from the group consisting of the promoter, 5'UTR, first exon, first intron, second exon, second intron, third exon, third intron, fourth exon, fourth intron, fifth exon, fifth intron, sixth exon, sixth intron, seventh exon, seventh intron, eighth exon, 3'UTR, terminator, and any combination thereof. In another embodiment, the mutant pmt allele contains a mutation in a PMT genome sequence region listed in Tables 1D to 1H.

[0045] In another embodiment, the mutant pmt allele comprises one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof. In one embodiment, the mutant pmt allele is a null allele or a knockout allele.

[0046] In one embodiment, the mutant PMT allele results in one or more of the following: PMT protein cleavage, untranslatable PMT gene transcript, non-functional PMT protein, immature stop codon in the PMT gene, and any combination thereof.

[0047] In another embodiment, the mutant PMT allele comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the wild-type PMT gene.

[0048] In one embodiment, the pmt mutant includes a zygote state selected from the group consisting of homozygosity, heterozygosity, and heteroalleles. In another embodiment, the pmt mutant is homozygosity or heteroalleles in at least 1, 2, 3, 4, or 5 PMT genes. In one embodiment, the pmt mutant is homozygosity or heteroalleles in at least 4 PMT genes. In another embodiment, the pmt mutant is homozygosity or heteroalleles in all 5 PMT genes. In another embodiment, the pmt mutant includes mutations in PMT1a and PMT3.

[0049] In one embodiment, a tobacco plant can produce leaves containing nicotine levels selected from the group consisting of less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01% by dry weight.

[0050] In another embodiment, tobacco plants can produce leaves containing total alkaloid levels selected from the group consisting of less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2% by dry weight.

[0051] In a further embodiment, tobacco plants can produce dried leaves containing total TSNA levels of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 ppm.

[0052] In one embodiment, when dried, tobacco plants can produce leaves having USDA grade index values ​​selected from the group consisting of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another embodiment, when dried, tobacco plants can produce leaves having USDA grade index values ​​equivalent to those of a control plant when grown and dried under similar conditions, and the control plant shares essentially the same genetic background as tobacco plants, except for modifications. In yet another embodiment, when dried, tobacco plants can produce leaves having USDA grade index values ​​of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of the USDA grade index values ​​of a control plant when grown under similar conditions, and the control plant shares essentially the same genetic background as tobacco plants, except for modifications. In a further embodiment, tobacco plants, when dried, can produce leaves having USDA grade index values ​​of 65%-130%, 70%-130%, 75%-130%, 80%-130%, 85%-130%, 90%-130%, 95%-130%, 100%-130%, 105%-130%, 110%-130%, 115%-130%, or 120%-130% of the USDA grade index values ​​of the control plant. In a further embodiment, tobacco plants, when dried, can produce leaves having USDA grade index values ​​of 70%-125%, 75%-120%, 80%-115%, 85%-110%, or 90%-100% of the USDA grade index values ​​of the control plant.

[0053] In one embodiment, the tobacco plant, when grown under similar growth conditions, contains nicotine at levels less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of the control plant, and the control plant, apart from modifications, shares essentially the same genetic background as the tobacco plant.

[0054] In a further embodiment, the tobacco plant comprises one or more pmt mutant alleles and further comprises a transgene or mutation that directly suppresses the expression or activity of one or more genes encoding products selected from the group consisting of MPO, QPT, BBL, A622, aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, ornithine decarboxylase, arginine decarboxylase, nicotine uptake permease (NUP), and MATE transporter.

[0055] In one embodiment, the tobacco plant contains one or more pmt mutant alleles and further contains a mutation in the ERF gene at the Nic2 locus. In another embodiment, the tobacco plant further contains one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. See Shoji et al., Plant Cell, (10):3390-409 (2010); and Kajikawa et al., Plant physiol. 2017, 174:999-1011. In another embodiment, the tobacco plant further contains one or more mutations in ERF189, ERF115, or both.

[0056] In one embodiment, the tobacco plant contains one or more qpt mutant alleles and further includes a mutation in the ERF gene at the Nic1 locus (or Nic1b locus, as seen in PCT / US2019 / 013345 filed on January 11, 2019, published as WO / 2019 / 140297). See also WO / 2018 / 237107. In one embodiment, the tobacco plant further includes one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See Kajikawa et al., Plant physiol. 2017, 174:999-1011. In one embodiment, the tobacco plant further comprises one or more mutations in one or more, two or more, three or more, four or more, five or more, or all six genes selected from the group consisting of ERFnew, ERF199, ERF19, ERF29, ERF210, and ERF91L2.

[0057] In one embodiment, the disclosure further provides a PMT mutant tobacco plant or a portion thereof containing a nicotine level or total alkaloid level selected from the group consisting of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.025%, less than 0.01%, and less than 0.005%, wherein the tobacco plant, when dried, can produce leaves having USDA grade index values ​​of 50 or higher, 55 or higher, 60 or higher, 65 or higher, 70 or higher, 75 or higher, 80 or higher, 85 or higher, 90 or higher, and 95 or higher. In another embodiment, such a PMT mutant tobacco plant contains a nicotine level of less than 0.02% and, upon drying, can produce leaves with a USDA grade index value of 70 or higher. In yet another embodiment, such a tobacco plant contains a nicotine level of less than 0.01% and, upon drying, can produce leaves with a USDA grade index value of 70 or higher.

[0058] In one embodiment, the disclosure also provides a tobacco plant or a portion thereof containing a non-transgenic mutation, the non-transgenic mutation reducing the nicotine level or total alkaloid level of the tobacco plant to less than 1%, less than 2%, less than 5%, less than 8%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80% of the nicotine level of a control plant when grown under similar growth conditions, the tobacco plant can produce leaves having a USDA grade index value equivalent to that of a control plant when dried, the control plant sharing essentially the same genetic background as the tobacco plant excluding the non-transgenic mutation.

[0059] In one embodiment, the tobacco plant contains a pmt mutation introduced by an approach selected from a group consisting of random mutagenesis and targeted mutagenesis. In another embodiment, the pmt mutation is introduced by a targeted mutagenesis approach selected from a group consisting of meganucleases, zinc finger nucleases, TALENs, and CRISPR.

[0060] Unless otherwise specified, for tobacco plants, varieties, cultivars, or strains, the measured values ​​of alkaloid or nicotine levels (or chemical properties or characterizations of another leaf) or leaf grade index values ​​referred to herein refer to average measurements, including, for example, the average of multiple leaves from a single plant, or average measurements from a population of tobacco plants derived from a single variety, cultivar, or strain.

[0061] Unless otherwise specified, nicotine or alkaloid levels (or chemical properties or characterizations of another leaf) of tobacco plants are measured post-topping in pooled leaf samples collected from leaf numbers 3, 4, and 5 after topping. Whenever a comparison between leaves from two plants (e.g., a mutant plant and a control plant) is referred to as used herein, leaves from the same or equivalent leafing position(s) and developmental stage(s) are intended, and the comparison can demonstrate the effect of genotype differences rather than other factors. For example, leaf 3 of a wild-type control plant is intended as a reference point for comparison with leaf 3 of a pmt mutant plant. In one embodiment, a tobacco plant containing at least one pmt mutation is compared with a control tobacco plant of the same tobacco variety.

[0062] Nicotine or alkaloid levels of tobacco plants (or chemical properties or characterization of another leaf) can also be measured by alternative methods. In one embodiment, the nicotine or alkaloid levels of tobacco plants (or chemical properties or characterization of another leaf) are measured after flowering in the leaf with the highest level of nicotine or alkaloids (or chemical properties or characterization of another leaf). In one embodiment, the nicotine or alkaloid levels of tobacco plants are measured after flowering in leaf numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another embodiment, the nicotine level or alkaloid level of a tobacco plant (or the chemical properties or characterization of another leaf) is measured after flowering in two or more pools of consecutive leaf counts selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 leaves. In another embodiment, the nicotine level or alkaloid level of a tobacco plant (or the chemical properties or characterization of another leaf) is measured after flowering in leaves with a number of leaves selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30 leaves. In another embodiment, the nicotine level or alkaloid level of a tobacco plant (or the chemical properties or characterization of another leaf) is measured after flowering in a pool of two or more leaves with a number of leaves selected from the groups consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30. In another embodiment, the nicotine level or alkaloid level of a tobacco plant (or the chemical properties or characterization of another leaf) is measured after flowering in a pool of three or more leaves with a number of leaves selected from the groups consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30.

[0063] As used herein, leaf numbering is based on the position of the leaves on the tobacco stem, with leaf number 1 after flowering being the youngest leaf (upper part) and the highest leaf number being the oldest leaf (lower part).

[0064] The tobacco plant population or tobacco leaf collection used to determine average values ​​(e.g., alkaloid levels, nicotine levels, or leaf grading) can be of any size, e.g., 5, 10, 15, 20, 25, 30, 35, 40, or 50. Industry-recognized standard protocols are followed to determine the average values ​​or grading index values.

[0065] As used herein, “flower thinning” refers to the removal of the shoot apex, including the apical meristem (SAM), flowers, and up to several adjacent leaves, when the tobacco plant is nearing maturity and around the time of the start of reproductive growth. Typically, tobacco plants are flower thinned at the button stage (immediately after flowers begin to appear). For example, tobacco plants grown in a greenhouse or field can be flower thinned when 50% of the plants have at least one flower. Flower thinning of tobacco plants results in the loss of apical dominance and induces increased alkaloid production.

[0066] Unless otherwise indicated, nicotine or alkaloid levels of tobacco plants (or chemical properties or characterization of another leaf) are measured two weeks after flowering. Alternatively, other time points may be used. In one embodiment, nicotine or alkaloid levels of tobacco plants (or chemical properties or characterization of another leaf) are measured approximately one, two, three, four, or five weeks after flowering. In another embodiment, nicotine, alkaloid, or polyamine levels of tobacco plants (or chemical properties or characterization of another leaf) are measured approximately three, five, seven, ten, twelfth, fourteenth, seventeenth, nineteenth, or twenty-one days after flowering.

[0067] As used herein, “similar growth conditions” or “equivalent growth conditions” refers to similar environmental conditions and / or agricultural practices used to grow and make meaningful comparisons between two or more plant genotypes, where neither the environmental conditions nor the agricultural practices contribute to nor explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (e.g., nitrogen and phosphorus). Agricultural practices include, for example, seeding, clipping, undercutting, transplanting, topping, and suckering. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70–103.

[0068] Alkaloids are complex nitrogen-containing compounds that occur naturally in plants and have pharmacological effects in humans and animals. Nicotine is the major natural alkaloid in commercially available cigarette tobacco, accounting for approximately 90% of the alkaloid content in Nicotiana tabacum. Other major alkaloids in tobacco include cotinine, nornicotine, myosmin, nicotine, anabasine, and anatabine. Trace amounts of tobacco alkaloids include nicotine-n-oxide, N-methylanatabine, N-methylanabasine, pseudooxynicotine, 2,3-dipyridyl, and others.

[0069] Alkaloid levels can be assayed by methods known in the art, such as quantification based on gas-liquid chromatography, high-performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay. For example, nicotinic alkaloid levels can be measured by GC-FID based on CORESTA Recommended Method No. 7, 1987 and ISO standard (ISO TC 126N 394 E). For a method using gas-liquid chromatography equipped with a capillary column and FID detector, see Hibi et al., Plant Physiology 100:826-35 (1992).

[0070] Unless otherwise specified, alkaloid and nicotine levels are measured using methods defined in CORESTA Method 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography, February 2005, and Federal Register Vol. 64, No. 55, published March 23, 1999 (and amended Vol. 74, No. 4, January 7, 2009), the Centers for Disease Control and Prevention Protocol for the Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products. Alternatively, total alkaloids in tobacco can be measured using the segment flow colorimetric method adapted by Skalar Instrument Co (West Chester, PA) and developed for the analysis of tobacco samples as described in Collins et al., Tobacco Science 13:79-81 (1969). In short, tobacco samples can be dried, ground, and extracted before analysis of total alkaloids and reducing sugars. The method then uses acetic acid / methanol / water extract and charcoal for decolorization. The determination of total alkaloids is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines, forming a colored complex measured at 460 nm. Unless otherwise specified, the total alkaloid levels or nicotine levels shown herein are based on dry weight (e.g., percentage of total alkaloids or percentage of nicotine).

[0071] In one embodiment, tobacco plants are present in approximately 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2% by dry weight. This includes mean nicotine levels or total alkaloid levels selected from the group consisting of %, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9%. In another aspect, tobacco plants, on a dry weight basis, are approximately 0.01%~0.02%, 0.02%~0.05%, 0.05%~0.75%, 0.75%~0.1%, 0.1%~0.15%, 0.15%~0.2%, 0.2%~0.3%, 0.3%~0.35%, 0.35%~0.4%, 0.4%~0.5%, 0.5%~0.6%, 0.6%~0.7%, 0.7%~0.8%, 0.8%~0.9%, 0.9%~1%, 1%~1.1%, 1.1%~1.2%, 1.2%~1.3%, 1.3%~1.4%, 1.4%~1.5%, 1.5%~1 This includes average nicotine levels or total alkaloid levels selected from the group consisting of 0.6%, 1.6%~1.7%, 1.7%~1.8%, 1.8%~1.9%, 1.9%~2%, 2%~2.1%, 2.1%~2.2%, 2.2%~2.3%, 2.3%~2.4%, 2.4%~2.5%, 2.5%~2.6%, 2.6%~2.7%, 2.7%~2.8%, 2.8%~2.9%, 2.9%~3%, 3%~3.1%, 3.1%~3.2%, 3.2%~3.3%, 3.3%~3.4%, 3.4%~3.5%, and 3.5%~3.6%. In a further embodiment, the tobacco plant contains an average nicotine level or total alkaloid level selected from the group consisting of approximately 0.01%–0.1%, 0.02%–0.2%, 0.03%–0.3%, 0.04%–0.4%, 0.05%–0.5%, 0.75%–1%, 0.1%–1.5%, 0.15%–2%, 0.2%–3%, and 0.3%–3.5% on a dry weight basis.

[0072] This disclosure also provides tobacco plants having altered nicotine levels that do not adversely affect other tobacco traits, such as leaf grade index values. In one embodiment, low-nicotine or nicotine-free tobacco varieties provide commercially acceptable grades of dried tobacco. Tobacco grades are evaluated based on factors including, but not limited to, leaf placement, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, resilience, luster (related to leaf strength and color depth and shine), hygroscopicity (the ability of tobacco leaves to absorb and retain ambient moisture), and green hue or color cast. Leaf grades can be determined, for example, using official standard grades published by the Agricultural Marketing Service of the United States Department of Agriculture (7U.SC §511).For example, the official standard grades for Burley Tobacco (US Type 31 and Foreign Type 93) effective November 5, 1990 (55F.R.40645), the official standard grades for Flue-Cured Tobacco (US Types 11, 12, 13, 14 and Foreign Type 92) effective March 27, 1989 (54F.R.7925), the official standard grades for Pennsylvania Seedleaf Tobacco (US Type 41) effective January 8, 1965 (29F.R.16854), and Ohio Cigar-Leaf Tobacco effective December 8, 1963. See the official standard grades for Tobacco (US Types 42, 43, and 44) ​​(28F.R.11719 and 28F.R.11926), the official standard grades for Wisconsin Cigar-Binder Tobacco (US Types 54 and 55) (34F.R.17061) effective November 20, 1969, the official standard grades for Wisconsin Cigar-Binder Tobacco (US Types 54 and 55) (34F.R.17061) effective November 20, 1969, and the official standard grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (US Type 62) effective April 1971. USDA grade index values ​​can be determined according to industry-recognized grade indexes. See, for example, Bowman et al, Tobacco Science, 32:39-40 (1988), Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index), and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the above references are relied upon in their entirety).In one embodiment, the USDA grading index is a numerical representation of the federal grading system, ranging from 0 to 100, and is a weighted average of all leaf positions. A higher grading index indicates higher quality. Alternatively, leaf grading can be determined via hyperspectral imaging. See, for example, WO2011 / 027315 (published March 10, 2011, whose entirety is incorporated by inference).

[0073] In one embodiment, the tobacco plants provided herein contain, when grown under similar growth conditions, one or more tobacco aromatic compounds at similar levels compared to a control tobacco plant. In another embodiment, the tobacco plants provided herein contain, when grown under similar growth conditions, one or more tobacco aromatic compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, rhabdenoids, sembrenoids, sugar esters, and reducing sugars at similar levels compared to a control tobacco plant.

[0074] As used herein, tobacco aromatic compounds are compounds associated with the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, sembrenoids and rhabdenoids, diterpenes, and sugar esters. The concentrations of tobacco aromatic compounds can be measured by any known metabolite profiling method in the art, including, but not limited to, gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance spectroscopy, and liquid chromatography-mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

[0075] As used herein, “reducing sugars” is any sugar (monosaccharide or polysaccharide) having free or potentially free aldehyde or ketone groups. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing the pH of the smoke and effectively reducing the amount of “free” unprotonated nicotine. Reducing sugars balances the flavor of the smoke, for example, by altering the sensory effects of nicotine and other tobacco alkaloids. The inverse relationship between sugar content and alkaloid content has been reported between tobacco varieties, within the same variety, and within the same plant strain due to growing conditions. The reduction of sugar levels can be measured using the segment flow colorimetric method adapted by Skalar Instrument Co (West Chester, PA) and developed for the analysis of tobacco samples, as described in Davis, Tobacco Science 20:139-144 (1976). For example, the sample is dialyzed against a sodium carbonate solution. Neocuproine copper is added to the sample and the solution is heated. Neocuproine copper chelate is reduced in the presence of sugar, resulting in a colored complex that can be measured at 460 nm.

[0076] In one embodiment, the tobacco plant contains one or more non-naturally occurring mutant alleles at one or more PMT loci, reducing or eliminating the PMT enzyme activity from one or more PMT loci. In one embodiment, these mutant alleles result in lower nicotine levels. The mutant PMT alleles can be introduced by any method known in the art, including random mutagenesis or targeted mutagenesis approaches.

[0077] Such mutagenesis methods include, but are not limited to, seed treatment with ethyl methylsulfate (EMS) (Hildering and Verkerk, In, The use of induced mutations in plant breeding. Pergamon press, pp 317-320, 1965), UV irradiation, X-ray and fast neutron irradiation (see, e.g., Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971, and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3rd sup.rd ed), 1987), transposon tagging (Fedoroff et al., 1984, U.S. Patent Nos. 4,732,856 and 5,013,658), and T-DNA insertion (Hoekema et al., 1983; U.S. Patent No. 5,149,645). EMS-induced mutagenesis consists of random point mutations chemically induced across the length of the genome. Fast neutron mutagenesis involves exposing seeds to neutron irradiation, causing large deletions through double-strand DNA disruption. Transposon tagging involves inserting transposons into endogenous genes to reduce or eliminate gene expression. Types of mutations that may be present in tobacco genes include, for example, point mutations, deletions, insertions, duplications, and inversions. Preferably, such mutations are located in the coding region of tobacco genes, but mutations in the promoter region, introns, or untranslated regions of tobacco genes may also be desirable.

[0078] In addition, rapid and automatable methods for screening for chemically induced mutations, TILLING (targeted local disruption of the genome), using denatured HPLC or selective endonuclease digestion of selected PCR products are also applicable to this disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457. Mutations that affect gene expression or interfere with gene function can be determined using methods well known in the art. Insertion mutations in gene exons usually result in null mutants. Mutations in conserved residues may be particularly effective in inhibiting protein function. In one embodiment, a tobacco plant contains a nonsense (e.g., stop codon) mutation in one or more PMT genes described herein.

[0079] When identifying mutations, it is important to understand that the endogenous reference DNA sequence should be derived from the same tobacco variety. For example, if a modified tobacco plant containing a mutation is derived from variety TN90, the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if a modified tobacco cell containing a mutation is a TN90 cell, the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from tobacco cells derived from a different tobacco variety (e.g., K326).

[0080] In one embodiment, the disclosure also provides a tobacco strain having altered nicotine levels while maintaining commercially acceptable leaf quality. This strain can be produced by introducing mutations into one or more PMT genes via precise genomic manipulation techniques, such as activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly arranged short palindromic sequence repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and combinations thereof (see, for example, U.S. Patent Application Publication No. 2017 / 0233756). See, for example, Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013).

[0081] Screening and selection of mutagenic tobacco plants can be carried out by any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern spectroscopy, PCR amplification for the detection of polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for the detection of RNA transcripts, Sanger sequencing, next-generation sequencing techniques (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for the detection of enzymatic or ribozyme activity of polypeptides and polynucleotides, as well as protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-linked immunoassays for the detection of polypeptides. Other techniques such as in-situ hybridization, enzyme staining, and immunostaining can also be used to detect the presence or expression of polypeptides and / or polynucleotides. All methods for performing the cited techniques are known.

[0082] In one embodiment, the tobacco plant or plant genome provided herein is mutated or edited by a nuclease selected from the group consisting of meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas9 nuclease, CRISPR / Cpf1 nuclease, or CRISPR / Csm1 nuclease.

[0083] As used herein, “editing” or “genome editing” refers to the targeted 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 the removal or substitution of an endogenous plant genome nucleic acid sequence. In one embodiment, the edited nucleic acid sequence provided has sequence identity with the endogenous nucleic acid sequence of at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75%. In one embodiment, the provided edited nucleic acid sequence has at least 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, or at least 75% sequence identity with a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs. 11–15.

[0084] Meganucleases, ZFNs, TALENs, CRISPR / Cas9, CRISPR / Csm1, and CRISPR / Cpf1 induce double-strand DNA breaks at target sites in the genome sequence, which are then repaired by the natural processes of homologous recombination (HR) or non-homologous end joining (NHEJ). Sequence alterations then occur at the break site and may include deletions or insertions resulting in gene disruption in the case of NHEJ, or incorporation of a donor nucleic acid sequence via HR. In one embodiment, the provided method comprises editing a plant genome using the provided nuclease, thereby mutating at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten nucleotides in the plant genome via HR with a donor polynucleotide. In one embodiment, the provided mutations are caused by genome editing using the nuclease. In another embodiment, the provided mutations are caused by non-homologous end joining or homologous recombination.

[0085] Meganucleases commonly identified in microorganisms are intrinsic enzymes with high activity and long recognition sequences (>14 bp) that result in site-specific digestion of target DNA. Manipulated versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14–40 bp). Manipulating meganucleases can be more challenging than manipulating ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined within a single domain. Specialized methods of mutagenesis and high-throughput screening are used to create novel meganuclease variants that recognize specific sequences and possess improved nuclease activity.

[0086] ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of a FokI restriction endonuclease. ZFNs can be designed to cleave double-stranded DNA of virtually any length due to modifications of the zinc finger DNA-binding domain. ZFNs form dimers from monomers consisting of a nonspecific DNA-cleavage domain of a FokI endonuclease fused to a zinc finger array engineered to bind to a target DNA sequence.

[0087] The DNA-binding domain of a ZFN typically consists of a 3-4 zinc finger array. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger ∞-helix, which contribute to site-specific binding to target DNA, can be modified and customized to fit specific target sequences. Other amino acids form a consensus skeleton to produce ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.

[0088] The FokI nuclease domain requires dimerization to cleave DNA, and therefore, two ZFNs with their C-terminal regions are needed to bind to the DNA strand opposite the cleavage site (separated at 5–7 bp). A ZFN monomer can cleave a target site if the two ZF binding sites are in a palindromic structure. As used herein, the term ZFN is broad and includes monomeric ZFNs that can cleave double-stranded DNA without the assistance of another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs that have been engineered to work together to cleave DNA at the same site.

[0089] Without being limited by any scientific theory, the DNA binding specificity of zinc finger domains can, in principle, be remanufactured using one of several methods; therefore, customized ZFNs can theoretically be constructed to target almost any gene sequence. Commonly available methods for manipulating zinc finger domains include context-dependent assembly (CoDA), oligomerized pool engineering (OPEN), and modular assembly.

[0090] TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA-binding domain to a FokI nuclease domain. When each member of a TALEN pair binds to a DNA site adjacent to the target site, the FokI monomer dimerizes, causing a double-strand DNA break at the target site. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave double-strand DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0091] Transcription activator-like effectors (TALEs) can be manipulated to bind to virtually any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. Xanthomonas pathogens secrete TALEs into host plant cells during infection. TALEs translocate to the nucleus and recognize and bind to specific DNA sequences in the promoter regions of specific genes in the host genome. TALEs have a central DNA-binding domain consisting of 13 to 28 repeats of a 33-34 amino acid monomer. The amino acids in each monomer are highly conserved, except for the hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeating variable duos (RVDs). The amino acid pairs of RVDs, NI, NG, HD, and NN, preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and the regulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequences and DNA recognition allowed for the manipulation of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0092] In addition to the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs, each having a DNA-binding domain specific to a target genomic site with appropriate orientation and spacing. Both the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites, are parameters for achieving high levels of activity.

[0093] The relationship between the amino acid sequence of the TALE-binding domain and DNA recognition enables the design of constructible proteins. TALE constructs can be designed using software programs such as DNAWorks. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al., Nucleic Acids Research (2012) 40:W117-122, Cermak et al., Nucleic Acids Research (2011) 39:e82, and tale-nt.cac.cornell.edu / about.

[0094] The CRISPR / Cas9, CRISPR / Csm1, or CRISPR / Cpf1 systems are alternatives to the FokI-based methods ZFN and TALEN. CRISPR systems are based on RNA-inducible, engineered nucleases that utilize complementary base pairing to recognize DNA sequences at target sites.

[0095] The CRISPR / Cas9, CRISPR / Csm1, and CRISPR / Cpf1 systems are part of the adaptive immune systems of bacteria and archaea, protecting them from invading nucleic acids such as viruses by cleaving foreign DNA in a sequence-dependent manner. Immunity is acquired by the incorporation of a short fragment of invading DNA known as a spacer between two adjacent repeats at the proximal end of the CRISPR locus. The CRISPR array containing the spacer is transcribed during subsequent encounter with the invading DNA and processed into a small interfering CRISPR RNA (crRNA) about 40 nt long, which, in combination with transactivating CRISPR RNA (tracrRNA), activates and induces the Cas9 nuclease. This cleaves homologous double-stranded DNA sequences known as protospacers in the invading DNA. A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which typically has the sequence 5-NGG-3 and less frequently has NAG. Specificity is provided by a so-called "seed sequence" approximately 12 base pairs upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 and Csm1 act in a similar manner to Cas9, but Cpf1 and Csm1 do not require tracrRNA.

[0096] In yet another embodiment, the tobacco plants provided herein include one or more pmt mutations and further include one or more mutations conferring a reduced amount of nornicotine at one or more loci encoding nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) compared to a control plant lacking one or more mutations at one or more loci encoding nicotine demethylase (see U.S. Patents 8,319,011, 8,124,851, 9,187,759, 9,228,194, 9,228,195, and 9,247,706). In one embodiment, the described tobacco plants further include reduced nicotine demethylase activity compared to a control plant when grown and dried under equivalent conditions.

[0097] In one embodiment, the pmt mutant tobacco plant further includes a mutation that, when grown and processed under comparable conditions, can produce leaves containing anabasine levels lower than those of leaves from a wild-type control tobacco plant. In another embodiment, the pmt mutant tobacco plant further includes a mutation that, when grown and processed under comparable conditions, can produce leaves containing anabasine levels lower than 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of those of leaves from a wild-type control tobacco plant.

[0098] In one embodiment, the pmt mutant tobacco plant includes further mutations that, when grown and processed under comparable conditions, produce leaves containing anatabin levels reduced by more than twofold compared to leaves from a control tobacco plant. In another embodiment, the pmt mutant tobacco plant includes further mutations that, when grown and processed under comparable conditions, produce leaves containing anatabin levels reduced by more than three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteenfold compared to leaves from a wild-type control tobacco plant. In one embodiment, the mutations that provide lower levels of anatabin are those described in U.S. Patent Publication Nos. 2014 / 0283165 and 2016 / 0010103. In another embodiment, the pmt mutant further includes mutations in the quinolate phosphoribosyltransferase (QPT) or quinolate synthase (QS) gene. In a further embodiment, the pmt mutant plant further includes transgenes or mutations that suppress the expression or activity of the QPT or QS gene.

[0099] In one embodiment, the PMT mutant tobacco plant further includes a mutation that, when grown and processed under equivalent conditions, provides nornicotine levels lower than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or 35% of the nornicotine levels of leaves from a wild-type control tobacco plant.

[0100] In one embodiment, pmt mutant tobacco plants can produce dried leaves containing total N-nitrosonornicotine (NNN) levels of less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05 ppm.

[0101] In another embodiment, pmt mutant tobacco plants can produce dried leaves containing total NNN levels of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 parts per million (ppm).

[0102] In one embodiment, the pmt mutant tobacco plant can produce dried leaves containing total nicotine-derived nitrosamine ketone (NNK) levels of less than 2, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.15, less than 0.1, or less than 0.05 ppm.

[0103] In another embodiment, pmt mutant tobacco plants can produce dried leaves containing total NNK levels of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 ppm.

[0104] In one embodiment, the PMT mutant tobacco plant further comprises a mutation or transgene that provides one or more increased levels of antioxidants. In another embodiment, the PMT mutant tobacco plant further comprises a gene modification in an endogenous gene, further comprising one or more increased levels of antioxidants in the dried leaves compared to a control dried tobacco leaf lacking the gene modification, and the endogenous gene encodes an enzyme for antioxidant biosynthesis, an antioxidant regulatory transcription factor, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In yet another embodiment, the PMT mutant tobacco plant further comprises a transgene, further comprising one or more increased levels of antioxidants in the dried leaves compared to a control dried tobacco leaf lacking the transgene, and the transgene encodes or directly modulates an enzyme for antioxidant biosynthesis, an antioxidant regulatory transcription factor, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In one embodiment, the pmt mutant tobacco plant further comprises a transgene or cisgenic construct expressing one or more genes selected from the group consisting of AtPAP1, NtAN2, NtAN1, NtJAF13, NtMyb3, colismyic acid mutase, and allogenic acid dehydrotase (ADT). In another embodiment, the pmt mutant tobacco plant further comprises one or more transgenes or genetic modifications for increasing antioxidants or decreasing one or more TSNAs, as described in WIPO Publication 2018 / 067985 or U.S. Publication 2018 / 0119163.

[0105] In one embodiment, the tobacco plant described is a modified tobacco plant. As used herein, "modified in the context of a plant" means a plant that has been modified for a particular purpose and that includes genetic alterations introduced beyond natural polymorphism.

[0106] In one embodiment, the tobacco plant described is a cisgenic plant. As used herein, “cisgenesis” or “cisgenic” refers to a genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoter, donor nucleic acid, select gene) are of plant origin only (i.e., no non-plant-derived components are used). In one embodiment, the plant, plant cell, or plant genome provided is cisgenic. The provided cisgenic plant, plant cell, and plant genome may lead to a ready-to-use tobacco strain. In another embodiment, the provided tobacco plant does not contain non-tobacco genetic material or sequences.

[0107] As used herein, “gene expression” or gene expression refers to the biosynthesis or production of a gene product, including the transcription and / or translation of the gene product.

[0108] In one embodiment, the provided tobacco plant comprises one or more pmt mutations and further comprises reduced expression or activity of one or more genes involved in nicotine biosynthesis or nicotine transport. Genes involved in nicotine biosynthesis include, but are not limited to, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosyl anthranilate isomerase (PRAI), quinolinate phosphoribosyltransferase (QPT), and S-adenosylmethionine synthase (SAMS). Nicotine synthases that catalyze the condensation step between nicotinic acid derivatives and methylpyrrolinium cations have two candidate genes proposed (A622 and NBB1), but they remain undetermined. See US2007 / 0240728A1 and US2008 / 0120737A1. A622 encodes an isoflavone reductase-like protein. In addition, several transporters may be involved in nicotine transport. A transporter gene named MATE has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)).

[0109] In one embodiment, the provided tobacco plant contains one or more pmt mutations and further comprises reduced levels of mRNA, protein, or both of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1 compared to a control tobacco plant. In another embodiment, the provided tobacco plant contains one or more pmt mutations and further comprises a transgene that directly represses the expression of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In yet another embodiment, the provided tobacco plant contains one or more pmt mutations and further comprises a transgene or mutation that represses the expression or activity of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1.

[0110] In one embodiment, the tobacco plant provided is derived from a tobacco variety selected from the group consisting of hot-air dried tobacco, air-dried tobacco, dark-colored air-dried tobacco, dark-colored open-fire dried tobacco, Galpao tobacco, and Oriental tobacco. In another embodiment, the tobacco plant provided is derived from a tobacco variety selected from the group consisting of Burley tobacco, Maryland tobacco, and dark-colored tobacco.

[0111] In one embodiment, the tobacco plant provided is either the background of hot-air dried tobacco or exhibits one or more of the characteristics of hot-air dried tobacco described herein. Hot-air dried tobacco (also known as Virginia tobacco or light tobacco) accounts for approximately 40% of the world's tobacco production. Hot-air dried tobacco is often referred to as "light tobacco" because it turns from golden to deep orange during drying. Hot-air dried tobacco has a light and distinctive aroma and taste. Hot-air dried tobacco is generally high in sugar and low in oil. The main growing countries of hot-air dried tobacco are Argentina, Brazil, China, India, Tanzania, and the United States. In one embodiment, the low-alkaloid or low-nicotine tobacco plants or seeds provided are based on hot-air dried tobacco selected from the group consisting of CC13, CC27, CC33, CC37, CC65, CC67, CC700, GF318, GL338, GL368, GL939, K346, K399, K326, NC102, NC196, NC291, NC297, NC299, NC471, NC55, NC606, NC71, NC72, NC92, PVH1118, PVH1452, PVH2110, SPEIGHT168, SPEIGHT220, SPEIGHT225, SPEIGHT227, SPEIGHT236, and any variety essentially derived from any one of the aforementioned varieties.In another embodiment, the low-alkaloid or low-nicotine tobacco plants or seeds offered include Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL939, K149, K326, K340, K346, K358, K394, K399, K730, NC27NF, NC37NF, NC55, NC60, NC71, NC72, NC82, NC95, NC297, NC606, NC729, NC2326, McNair 373, McNair 944, Ox207, Ox414NF, and Reams. The background is hot-air dried tobacco selected from the group consisting of 126, Reems 713, Reems 744, RG8, RG11, RG13, RG17, RG22, RG81, RGH4, RGH51, Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108, Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va 116, Va 182, and any variety essentially derived from any one of the aforementioned varieties. See WO2004 / 041006A1. In a further embodiment, a low-alkaloid or low-nicotine tobacco plant, seed, hybrid, variety, or strain is hot-air dried on any hot-air dried background selected from the group consisting of K326, K346, and NC196.

[0112] In one embodiment, the tobacco plants provided are the background for air-dried tobacco or exhibit one or more of the characteristics of air-dried tobacco described herein. Air-dried tobaccos include Burley, Maryland, and dark tobaccos. A common factor is that drying does not primarily involve the use of artificial heat and humidity sources. Burley tobacco is light to dark brown, high in oil, and low in sugar. Burley tobacco is air-dried in sheds. Major growing countries for Burley are Argentina, Brazil, Italy, Malawi, and the United States. Maryland tobacco is very fluffy, has good burning characteristics, is low in nicotine, and has a natural aroma. Major growing countries for Maryland include the United States and Italy. In one embodiment, the low-alkaloid or low-nicotine tobacco plants or seeds provided are based on Burley tobacco selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC2, NC3, NC4, NC5, NC2000, TN86, TN90, TN97, R610, R630, R711, R712, NCBH129, Bu21×Ky10, HB04P, Ky14×L8, Kt200, Newton 98, Pedigo 561, Pf561, and Va509. In a further embodiment, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or strains are on any Burley background selected from the group consisting of TN90, KT209, KT206, KT212, and HB4488. In another embodiment, the offered low-alkaloid or low-nicotine tobacco plants or seeds are on a Maryland tobacco background selected from the group consisting of Md10, Md40, Md201, Md609, Md872, and Md341.

[0113] In one embodiment, the tobacco plants provided are in the background of dark air-dried tobacco or exhibit the characteristics of one or more dark air-dried tobaccos described herein. Dark air-dried tobaccos are distinguished from other types primarily by their drying process, which gives them a medium to dark brown color and a distinctive aroma. Dark air-dried tobaccos are primarily used for chewing tobacco and snuff. In one embodiment, low-alkaloid or low-nicotine tobacco plants or seeds are provided in the background of dark air-dried tobaccos selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One sucker, Green River, Virginia sun-cured, and Paraguan Passado.

[0114] In one embodiment, the tobacco plant provided is the background for dark open-fire dried tobacco or exhibits the characteristics of one or more dark open-fire dried tobaccos described herein. Dark open-fire dried tobacco is generally dried over a low-burning bonfire on the floor of a closed drying shed. Its leaves have a low sugar content but a high nicotine content. Dark open-fire dried tobacco is used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-flavored cigars. The main growing regions for dark open-fire dried tobacco are Tennessee, Kentucky, and Virginia in the United States. In one embodiment, the low-alkaloid or low-nicotine tobacco plants or seeds provided are based on dark, open-burn dried tobacco 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, DT508, DT518, DT592, KY171, DF911, DF485, TND94, TND950, VA309, and VA359.

[0115] In one embodiment, the tobacco plant provided is based on oriental tobacco or exhibits one or more characteristics of oriental tobacco as described herein. Oriental tobacco is also known as Greek tobacco, aromatic tobacco, and Turkish tobacco, due to the fact that it is typically cultivated in the Eastern Mediterranean region, including Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leaf size, as well as its distinctive aromatic properties, which are characteristic of oriental varieties today, are the result of the plant's adaptation to poor soil and stressful climatic conditions cultivated over the past centuries. In one embodiment, the low-alkaloid or low-nicotine tobacco plants or seeds provided include Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, and Addu. The background of Oriental tobacco is selected from a group consisting of Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any variety that is essentially derived from any one of the aforementioned varieties.

[0116] In one embodiment, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or strains include BU64, CC101, CC200, CC27, CC301, CC400, CC500, CC600, CC700, CC800, CC900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU263, DF911, Galpao Tobacco, GL26H, GL350, GL600, GL737, GL939, GL973, HB04P, K149, K326, K346, K358, K394, K399, K730, KDH959, KT200, KT204LC, KY10, KY14, KY160, KY17, KY171, KY907, KY907LC, KTY14xL8LC, Little Crittenden, McNair373, McNair944, msKY14xL8, Narrow Leaf Mador, NC100, NC102, NC2000, NC291, NC297, NC299, NC3, NC4, NC5, NC6, NC7, NC606, NC71, NC72, NC810, NCBH129, NC2002, Neal Smith MadorMadole), OXFORD207, "Perique" cigarettes, PVH03, PVH09, PVH19, PVH50, PVH51, R610, R630, R7-11, R7-12, RG17, RG81, RGH51, RGH4, RGH51, RS1410, Speight168, Speight172, Speight ight179, Speight210, Speight220, Speight225, Speight227, Speight234, SpeightG-28, SpeightG-70, SpeightH-6, SpeightH20, SpeightNF3, TI1406, TI1269, TN86, TN86LC, TN9 It is essentially derived from or has a genetic background to any commercially available tobacco variety produced by standard tobacco breeding techniques known in the art, such as 0, TN97, TN97LC, TND94, TND950, TR (Tom Rosson) Madore, VA309 or VA359, Maryland609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, Shirey LC, or any other commercially available tobacco variety produced by standard tobacco breeding techniques known in the art.

[0117] All specific varieties of the aforementioned dark-colored air-dried, Burley, Maryland, dark-colored open-fire-dried, or Oriental varieties are listed for illustrative purposes only. Any additional dark-colored air-dried, Burley, Maryland, dark-colored open-fire-dried, oriental varieties are also considered in this application.

[0118] Also provided are populations of tobacco plants as described. In one embodiment, the tobacco plant population has a planting density of approximately 5,000 to 8,000, 5,000 to 7,600, 5,000 to 7,200, 5,000 to 6,800, 5,000 to 6,400, 5,000 to 6,000, 5,000 to 5,600, 5,000 to 5,200, 5,200 to 8,000, 5,600 to 8,000, 6,000 to 8,000, 6,400 to 8,000, 6,800 to 8,000, 7,200 to 8,000, or 7,600 to 8,000 plants per acre. In another embodiment, the tobacco plant population is found in soils with low to moderate fertility.

[0119] Also provided are containers for seeds derived from the tobacco plant described herein. The tobacco seed containers of this disclosure may contain any number, weight, or volume of seeds. For example, a container may contain about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, a container may contain about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds, or more seeds. The tobacco seed container may be any container available in the art. In non-limiting examples, a container may be a box, bag, packet, pouch, tape roll, tube, or bottle.

[0120] Dried tobacco materials made from tobacco plants with low alkaloid or low nicotine levels as described are also provided. Furthermore, dried tobacco materials made from tobacco plants with higher levels of total alkaloid or nicotine are also provided.

[0121] "Drying" is an aging process that reduces moisture, leads to the breakdown of chlorophyll, turns tobacco leaves golden, and converts starch into sugar. Therefore, dried tobacco has a higher reducing sugar content and a lower starch content compared to harvested green leaves. In one embodiment, the provided green leaf tobacco may be dried using conventional means, e.g., hot air drying, barn drying, open flame drying, air drying, or sun drying. For a description of different types of drying methods, see, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Dried tobacco is usually aged for several years (e.g., 2-5 years) under compression conditions in a wooden drum (e.g., hogshead) or cardboard box, with a moisture content ranging from 10% to about 25%. See, for example, U.S. Patent Nos. 4,516,590 and 5,372,149. Next, the dried and aged tobacco can be further processed. Further processing includes introducing steam at various temperatures, pasteurization, and adjusting the tobacco under vacuum, with or without fermentation. Fermentation is typically characterized by an initial high moisture content, exothermic reaction, and a 10-20% decrease in dry weight. See, for example, U.S. Patents 4,528,993, 4,660,577, 4,848,373, 5,372,149, U.S. Publication No. 2005 / 0178398, and Tso (1999, Tobacco, Production, Chemistry and Technology, Chapter 1, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Patents No. 4,528,993, No. 4,660,577, and No. 4,987,907. In one embodiment, the dried tobacco material of the Disclosure is sun-dried. In another embodiment, the dried tobacco material of the Disclosure is hot-air-dried, air-dried, or open-fire-dried.

[0122] The presence of mold on dried tobacco can significantly reduce the quality and marketability of the dried leaves (e.g., leaf grade). Mold growth is a common problem that can occur when high humidity (e.g., relative humidity above 70%) persists for extended periods at temperatures between approximately 10°C (50°F) and 32.2°C (90°F). Mold tends to be more common at higher temperatures.

[0123] Tobacco plants, varieties, and strains provided herein that include mutant alleles in one or more PMT genes, two or more PMT genes, three or more PMT genes, four or more PMT genes, or five PMT genes exhibit reduced fungal infection compared to the low-alkaloid tobacco variety LA Burley 21 (LA BU21). Similarly, tobacco plants, varieties, and strains provided herein that include RNAi constructs that downregulate the expression or translation of one or more PMT genes, two or more PMT genes, three or more PMT genes, four or more PMT genes, or five PMT genes exhibit reduced fungal infection compared to the low-alkaloid tobacco variety LA Burley 21 (LA BU21).

[0124] LA BU21 is a tobacco strain with low total alkaloids produced by incorporating low alkaloid genes(s) derived from Cuban cigar varieties into Burley 21 through several backcrosses (Legg et al., Crop Science, 10:212 (1970)). It has approximately 0.2% total alkaloids (dry weight) compared to approximately 3.5% (dry weight) of its parent, Barley 21. LA BU21's leaf grade falls far below commercially acceptable standards.

[0125] In one embodiment, dried tobacco leaves containing the PMT1a mutant allele are free from observable fungal infection. In another embodiment, dried tobacco leaves containing the PMT1b mutant allele are free from observable fungal infection. In another embodiment, dried tobacco leaves containing the PMT2 mutant allele are free from observable fungal infection. In another embodiment, dried tobacco leaves containing the PMT3 mutant allele are free from observable fungal infection. In another embodiment, dried tobacco leaves containing the PMT4 mutant allele are free from observable fungal infection. In yet another embodiment, dried tobacco leaves containing the PMT1a mutant allele, the PMT1b mutant allele, the PMT2 mutant allele, the PMT3 mutant allele, and the PMT4 mutant allele are free from observable fungal infection.

[0126] In one embodiment, dried tobacco leaves containing the pmt1a mutant allele contain reduced fungal infection compared to control dried tobacco leaves derived from various LA BU21 strains. In another embodiment, dried tobacco leaves containing the pmt1b mutant allele contain reduced fungal infection compared to control dried tobacco leaves derived from various LA BU21 strains. In another embodiment, dried tobacco leaves containing the pmt2 mutant allele contain reduced fungal infection compared to control dried tobacco leaves derived from various LA BU21 strains. In another embodiment, dried tobacco leaves containing the pmt3 mutant allele contain reduced fungal infection compared to control dried tobacco leaves derived from various LA BU21 strains. In yet another embodiment, dried tobacco leaves containing the pmt4 mutant allele contain reduced fungal infection compared to control dried tobacco leaves derived from various LA BU21 strains. In another embodiment, dried tobacco leaves containing mutant alleles of pmt1a, pmt1b, pmt2, pmt3, and pmt4 contain reduced fungal infection compared to control dried tobacco leaves derived from variety LA BU21.

[0127] In one embodiment, dried leaves from tobacco plants, varieties, or strains provided in any one of Tables 4A-4E, Table 10, or Table 14 are free from observable fungal infection. In another embodiment, dried leaves from tobacco plants, varieties, or strains provided in any one of Tables 4A-4E, Table 10, or Table 14 contain reduced fungal infection compared to control dried tobacco leaves from variety LA BU21.

[0128] In one embodiment, dried leaves from tobacco plants, varieties, or strains containing one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E are free from observable fungal infection. In another embodiment, dried leaves from tobacco plants, varieties, or strains containing one or more pmt mutations provided in any one of Tables 5A-5E and Tables 12A-12E contain reduced fungal infection compared to control dried leaves from variety LA BU21.

[0129] In one embodiment, dried leaves from a tobacco plant, variety, or strain containing the pmt1a mutant allele contain a higher leaf grade than control dried leaves from variety LA BU21. In another embodiment, dried leaves from a tobacco plant, variety, or strain containing the pmt1b mutant allele contain a higher leaf grade than control dried leaves from variety LA BU21. In another embodiment, dried leaves from a tobacco plant, variety, or strain containing the pmt2 mutant allele contain a higher leaf grade than control dried leaves from variety LA BU21. In another embodiment, dried leaves from a tobacco plant, variety, or strain containing the pmt3 mutant allele contain a higher leaf grade than control dried leaves from variety LA BU21. In another embodiment, dried leaves from a tobacco plant, variety, or strain containing the pmt4 mutant allele contain a higher leaf grade than control dried leaves from variety LA BU21. In another embodiment, dried tobacco leaves derived from a plant, variety, or strain containing the pmt1a mutant allele, pmt1b mutant allele, pmt2 mutant allele, pmt3 mutant allele, and pmt4 mutant allele contain a higher leaf grade than the control dried leaves of LA BU21.

[0130] In one embodiment, the dried leaves derived from tobacco plants, varieties, or strains provided in any one of Tables 4A to 4E, Table 10, or Table 14 contain a higher leaf grade than the control dried leaves derived from variety LA BU21.

[0131] In one embodiment, dried leaves from a tobacco plant, variety, or strain containing one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E contain a higher leaf grade than control dried leaves from variety LA BU21.

[0132] In one embodiment, “reduced fungal infection” refers to a reduced area of ​​infected leaves. In another embodiment, “reduced fungal infection” refers to a reduced number of surviving fungal spores on infected leaves. Standard methods for detecting and counting surviving spores are known and available in the art.

[0133] In one embodiment, reduced fungal infection includes a reduction of at least 1% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 2% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 3% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 4% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 5% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 10% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 15% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 20% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 25% in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a reduction of at least 30% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 35% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 40% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 50% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 60% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 70% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 75% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 80% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 90% in the infected leaf area compared to a control leaf. In one embodiment, reduced fungal infection includes a reduction of at least 95% in the infected leaf area compared to a control leaf. In another embodiment, reduced fungal infection includes a reduction of 100% in the infected leaf area compared to a control leaf.

[0134] In one embodiment, reduced fungal infection includes a 1% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 90% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 80% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 70% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 60% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 50% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 40% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 30% reduction in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a 1% to 20% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 1% to 10% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 10% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 20% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 30% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 40% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 50% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 60% to 100% reduction in infected leaf area compared to control leaves. In one embodiment, reduced fungal infection includes a 70% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes an 80% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 90% to 100% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 10% to 75% reduction in infected leaf area compared to control leaves. In another embodiment, reduced fungal infection includes a 25% to 75% reduction in infected leaf area compared to control leaves. In yet another embodiment, reduced fungal infection includes a 25% to 50% reduction in infected leaf area compared to control leaves.

[0135] In one embodiment, the molds that infect dried tobacco are those belonging to genera selected from the group consisting of Cladosporium, Penicillium, Alternaria, Aspergillus, and Mucor.

[0136] Tobacco products can be made using tobacco materials obtained from the tobacco strains, varieties, or hybrids of this disclosure. As used herein, “tobacco products” is defined as any product made from or derived from tobacco intended for human use or consumption.

[0137] The tobacco products offered include, but are not limited to, cigarette products (e.g., cigarettes and bidi-cigarettes), cigar products (e.g., cigar tobacco and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, molded parts, gels, consumable units, insoluble matrices, hollow shapes, recombined tobacco, and expanded tobacco. See, for example, U.S. Patent Publication US2006 / 0191548.

[0138] As used herein, “cigarette” refers to a tobacco product having a “rod” and a “filler.” The “rod” of a cigarette includes the cigarette paper, filter, plug wrap (used to contain the filter material), tip paper that holds the cigarette paper (including the filler) to the filter, and all adhesives that hold these components together. The “filler” includes (1) all tobacco, including but not limited to recomposed tobacco and expanded tobacco; (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other spices that may accompany tobacco rolled in cigarette paper); (3) casing; (4) flavorings; and (5) all other additives (mixed with tobacco and substitutes and rolled in cigarette).

[0139] As used herein, “reconstructed tobacco” refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap materials, processed into a sheet form, and cut into strips to resemble a cigarette. In addition to cost reduction, reconstructed tobacco is very important because it contributes to the taste of a cigarette through processing that uses a reaction between ammonia and sugar to produce flavor.

[0140] As used herein, “expanded tobacco” refers to a portion of tobacco filler that has been treated with gas expansion suitable for filling, thereby reducing its density and increasing its filling capacity. This reduces the weight of the tobacco used in the cigarette.

[0141] Furthermore, the plant-derived tobacco products of this disclosure include cigarettes and other smoking products, in particular smoking products that include filter elements, and rods of smoky material include dried tobacco in tobacco blends. In one embodiment, the tobacco products of this disclosure are selected from the group consisting of cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another embodiment, the tobacco products of this disclosure are smokeless tobacco products. Smokeless tobacco products do not burn and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snuff, and dry snuff. Chewing tobacco consists of coarsely divided tobacco leaves, typically packaged in a large pouch-like package and used for plugs or twists. Moist smokeless tobacco is moist, finely divided tobacco, supplied in loose or pouch form, typically packaged in a round tin, and used by adult tobacco consumers as a pinch or pouch, placed between the cheek and gums. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco used for oral or nasal use. In further embodiments, the tobacco products of the Disclosure are selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another embodiment, the tobacco products of the Disclosure are selected from the group consisting of electronically heated cigarettes, electronic cigarettes, and electronic evaporators.

[0142] In one embodiment, the tobacco product of the Disclosure may be a blended tobacco product. In another embodiment, the tobacco product of the Disclosure may be a low-nicotine tobacco product. In a further embodiment, the tobacco product of the Disclosure may contain nornicotine at a level of less than approximately 3 mg / g. For example, the nornicotine content in such products may be 3.0 mg / g, 2.5 mg / g, 2.0 mg / g, 1.5 mg / g, 1.0 mg / g, 750 μg / g, 500 pg / g, 250 pg / g, 100 pg / g, 75 pg / g, 50 pg / g, 25 pg / g, 10 pg / g, 7.0 pg / g, 5.0 pg / g, 4.0 pg / g, 2.0 pg / g, 1.0 pg / g, 0.5 pg / g, 0.4 pg / g, 0.2 pg / g, 0.1 pg / g, 0.05 pg / g, 0.01 pg / g, or undetectable.

[0143] In one embodiment, the provided dry tobacco material or tobacco product contains approximately 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, and 1.8% on a dry weight basis. This includes average nicotine levels or total alkaloid levels selected from the group consisting of 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9%. In another embodiment, the provided dry tobacco material or tobacco product is, on a dry weight basis, approximately 0.01%~0.02%, 0.02%~0.05%, 0.05%~0.75%, 0.75%~0.1%, 0.1%~0.15%, 0.15%~0.2%, 0.2%~0.3%, 0.3%~0.35%, 0.35%~0.4%, 0.4%~0.5%, 0.5%~0.6%, 0.6%~0.7%, 0.7%~0.8%, 0.8%~0.9%, 0.9%~1%, 1%~1.1%, 1.1%~1.2%, 1.2%~1.3%, 1.3%~1.4%, 1.4%~1.5% This includes average nicotine levels or total alkaloid levels selected from the groups consisting of %, 1.5%~1.6%, 1.6%~1.7%, 1.7%~1.8%, 1.8%~1.9%, 1.9%~2%, 2%~2.1%, 2.1%~2.2%, 2.2%~2.3%, 2.3%~2.4%, 2.4%~2.5%, 2.5%~2.6%, 2.6%~2.7%, 2.7%~2.8%, 2.8%~2.9%, 2.9%~3%, 3%~3.1%, 3.1%~3.2%, 3.2%~3.3%, 3.3%~3.4%, 3.4%~3.5%, and 3.5%~3.6%. In a further embodiment, the provided dry tobacco material or tobacco product contains an average nicotine level or total alkaloid level selected from the group consisting of approximately 0.01% to 0.1%, 0.02% to 0.2%, 0.03% to 0.3%, 0.04% to 0.4%, 0.05% to 0.5%, 0.75% to 1%, 0.1% to 1.5%, 0.15% to 2%, 0.2% to 3%, and 0.3% to 3.5% on a dry weight basis.

[0144] This disclosure also provides methods for breeding tobacco strains, cultivars, or varieties containing desired levels of total alkaloids or nicotine, e.g., low-nicotine or nicotine-free. Breeding can be carried out via any known procedure. DNA fingerprinting, SNP mapping, haplotype mapping, or similar techniques may be used in marker-assisted selection (MAS) breeding programs to introduce or breed desired traits or alleles into tobacco plants. For example, a breeder may use F1 hybrid plants to create segregated populations in the F2 or backcross generation, or further cross F1 hybrid plants with other donor plants having agronomically desirable genotypes. Plants in the F2 or backcross generation can be screened for desired agronomic traits or desired chemical characteristics using techniques known in the art or one of the techniques enumerated herein. Depending on the expected genetic pattern or the MAS technique used, self-pollination of selected plants may be performed before each cycle of backcrossing to aid in the identification of desired individual plants. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is restored. The recurrent parent in this disclosure may be a hot-air dried variety, a Burley variety, a dark air-dried variety, a dark fire-dried variety, or an Oriental variety. Other breeding techniques can be found, for example, in Wernsman, EA, and Rufty, RC 1987 Chapter Seventeen. Tobacco. Pages 669-698 Cultivar Development. Crop Species. WHFehr (ed.), MacMillan Publishing Go., Inc., New York, NY (these are incorporated herein by reference in their entirety).

[0145] The results of plant breeding programs using the tobacco plants described include useful strains, cultivars, varieties, offspring, inbred lines, and hybrids as disclosed herein. As used herein, the term “cultivar” refers to a group of plants that share certain characteristics that distinguish them from other plants of the same species. Cultivars are often, though not always, commercially traded. Cultivars are further characterized by very small overall variations among individuals within their cultivar, while possessing one or more characteristic traits. A “pure line” cultivar may be created by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. A cultivar may originate from essentially another strain or cultivar. As defined by the International Convention for the Protection of New Varieties of Plants (revised in Geneva on December 2, 1961, November 10, 1972, October 23, 1978, and March 19, 1991), a variety is "essentially derived" from the original variety if a) it is primarily derived from the original variety, or from a variety primarily derived from the original variety, while retaining the expression of essential characteristics resulting from the genotype or combination of genotypes of the original variety; b) it is clearly distinguishable from the original variety; and c) it is compatible with the original variety in the expression of essential characteristics resulting from the genotype or combination of genotypes of the original variety, except for differences resulting from derivative acts. Essentially derived varieties can be obtained, for example, by natural or induced mutants, somaclonal variants, variant individuals of the original variety derived from plants, backcrossing, or selection by transformation. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived are considered to have essentially the same genetic background. A "plant," as distinguished from a variety, most often refers to a group of plants used non-commercially, for example, in plant research. Plants typically show little overall variation among individuals with respect to one or more traits of interest, but some variation among individuals with respect to other traits.

[0146] In one embodiment, the disclosure provides a tobacco plant, variety, strain, or cell containing one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E.

[0147] In another embodiment, the Disclosure provides tobacco plants, varieties, strains, or cells derived from any tobacco plant, variety, or strain provided in any one of Tables 4A to 4E, Table 10, or Table 14.

[0148] In one embodiment, the disclosure provides tobacco strain 18GH203 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH341 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1678 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1680 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1804 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1898 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH207 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH342 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH343 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH348 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH349 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH355 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH359 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH64 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH682 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH692 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH697 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH922 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH957 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1808 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1810 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1886 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1888 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1889 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH189 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1893 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1901 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1902 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH3 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH125 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH208 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH403 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH414 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH434 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH436 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH437 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH449 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH706 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH709 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH710 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH716 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH729 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH731 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH752 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH756 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH768 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH771 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH776 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH800 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH818 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH10 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH1004 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH1033 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH132 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH134 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH217 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH456 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH457 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH460 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH465 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH71 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH830 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH831 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH836 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH841 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH974 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, this disclosure relates to tobacco stocks 18GH981 and F. The disclosure provides F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH994, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1905, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH128, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH130, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH131, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH133 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH136 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH216 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH227 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH5 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH6 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH65, and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH66, and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH69, and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH72 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH73 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH74 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH78 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH79 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH8 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH9 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1696 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1717 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1719 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1729 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1736 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1737 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1739 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1740 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 17GH1835 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1848 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1849 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1912 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1937 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1940 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1943 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1944 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH1051 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH22 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH34 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH473 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH49 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH50 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH848 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH850 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH851 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1699 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1708 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1722 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1724 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1725 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1845 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1846 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1847 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1911 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1912 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1915, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1918, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 17GH1928 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1932 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1933 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1936 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH20 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH28 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH31 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH47 and F1 or F2 tobacco plants, or derived therefrom. The disclosure provides male-sterile tobacco plants. In one embodiment, the disclosure provides tobacco strain 18GH51 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH52 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS107 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS106 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS115 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1809-13 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS111 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS112 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1678-60 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS131 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH709-01 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH709-08 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH414-11, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH414-19, and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 18GH437-04 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH437-08 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH437-32 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH437-39 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH449-26 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH449-33 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH125-48 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS102 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS103 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1719-30 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1740-36 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1698-22 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1700-13 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1702-17 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain 17GH1849-01 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1849-48 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 17GH1737-24 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS118 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS133 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS120 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH1108-07 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH2162 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS164 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS163 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS146 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS147 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS150, and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS151, and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS148, and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom.In one embodiment, the disclosure provides tobacco strain CS149 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS152 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS153 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS143 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH2169 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH2171 and F1 or F2 tobacco plants, or male sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS165 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain CS118 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom. In one embodiment, the disclosure provides tobacco strain 18GH2254-7 and F1 or F2 tobacco plants, or male-sterile tobacco plants derived therefrom.

[0149] In one embodiment, the present disclosure provides a method for introducing a low-nicotine trait into a tobacco variety, comprising: (a) crossing a first tobacco variety containing the low-nicotine trait with a second tobacco variety not containing the low-nicotine trait to produce one or more offspring tobacco plants; (b) genotyping one or more offspring tobacco plants for a pmt mutant allele selected from those listed in Tables 4A-4E, 5A-5E, 10, and 12A-12E; and (c) selecting offspring tobacco plants containing the pmt mutant allele. In another embodiment, these methods further include backcrossing the selected offspring tobacco plants with the second tobacco variety. In a further embodiment, these methods further include (d) crossing the selected offspring plants either by themselves or with the second tobacco variety to produce one or more further offspring tobacco plants; and (e) selecting further offspring tobacco plants containing the low-nicotine trait. In one embodiment, the selection in step (e) includes marker-assisted selection. In one embodiment, these methods generate a single gene transmutation containing a low nicotine trait. In one embodiment, these methods generate a single gene transmutation containing a pmt mutant allele. In one embodiment, the second tobacco variety is a superior variety. In another embodiment, the genotyping step of these methods involves one or more molecular marker assays. In another embodiment, genotyping may include polymorphic markers, including polymorphisms selected from the group consisting of single nucleotide polymorphisms (SNPs), insertions or deletions in DNA sequences (indels), simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RFLPs), and tagged SNPs.

[0150] As used herein, “locus” refers to a chromosomal locus or region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. A “locus” may refer to a corresponding locus or region shared by two homologous chromosomes. As used herein, “allele” refers to a gene or an alternative nucleic acid sequence at a particular locus (e.g., a nucleic acid sequence of the same gene or the same locus, but different from other alleles). Such an allele may be considered a variant if (i) the wild type, or (ii) one or more mutations or edits are present in the nucleic acid sequence of the mutant allele compared to the wild-type allele. A mutant allele of a gene may have reduced or eliminated gene activity or expression levels compared to the wild-type allele. In diploid organisms such as tobacco, the first allele may arise on one chromosome, and the second allele may arise at the same locus on a second homologous chromosome. If one allele at a locus on one chromosome of a plant is a mutant allele, and the other corresponding allele on the plant's homologous chromosome is wild-type, the plant is described as heterozygous for the mutant allele. However, if both alleles at a locus are mutant alleles, the plant is described as homozygous for the mutant allele. A plant that is homozygous for the mutant allele at a locus may contain the same mutant allele or different mutant alleles, whether heteroallelic or biallelic.

[0151] As used herein, “gene transfer” or “to transfer genes” refers to the transfer of a desired allele from one gene locus to another gene locus originating from a certain genetic background.

[0152] As used herein, “crossed” or “crossed” means producing offspring through fertilization (e.g., cells, seeds, or plants), and includes crossing between plants (sexual) and self-fertilization (self-pollination).

[0153] As used herein, “backcross” and “backcrossing” refer to the process of repeatedly crossing a descendant plant with one of its parents. In a backcross scheme, a “donor” parent refers to a parent plant that possesses the desired gene or locus to be introduced. A “recipient” parent (used once or more) or a “repeated” parent (used two or more times) refers to a parent plant into which the gene or locus is introduced. The initial cross produces the F1 generation. The term “BC1” refers to the second use of a repeating parent, “BC2” refers to the third use of a repeating parent, and so on. In one embodiment, backcrossing is repeated using the descendant individuals of each successive backcross generation so that they themselves are backcrossed to the genotype of the same parent.

[0154] As used herein, “single-gene transmuted” or “single-gene transmuted” refers to a plant developed using a plant breeding technique known as backcrossing, or through genetic engineering, in which a single gene is introduced into the variety via backcrossing or genetic engineering, and in addition, all of the desired morphological and physiological characteristics of the variety are essentially restored.

[0155] As used herein, “superior variety” means any variety resulting from breeding and selection for superior agricultural performance.

[0156] As used herein, “selecting” or “choosing” in the context of marker-assisted selection or breeding typically refers to the act of picking or choosing a desired individual from a population based on specific, predetermined criteria.

[0157] As used herein, the term “trait” refers to one or more detectable features of a cell or organism that may be influenced by genotype. Phenotypes may be observable with the naked eye or by any other means of evaluation known in the art, such as microscopy, biochemical analysis, genomic analysis, or specific disease resistance assays. In some cases, the phenotype is directly controlled by a single gene or locus, for example, a “single-gene trait.” In other cases, the phenotype is attributable to several genes.

[0158] As used herein, “marker assay” means a method for detecting polymorphism at a specific locus using a particular method, such as measuring at least one phenotype (e.g., seed color, flower color, or other visually detectable trait), restriction fragment length polymorphism (RFLP), single nucleotide elongation, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), randomly amplified polymorphic DNA (RAPD), microarray-based techniques, and nucleic acid sequencing techniques.

[0159] As used herein, “marker-assisted selection” (MAS) is a process in which a phenotype is selected based on a marker genotype. “Marker-assisted selection breeding” refers to a process of selecting a desired trait(s) from a plant(s) by detecting one or more nucleic acids from the plant(s), associating those nucleic acids with the desired trait(s), and then selecting plants or germplasm having one or more of those nucleic acids.

[0160] As used herein, “polymorphism” means the presence of one or more variations in a population. Polymorphism can manifest as variations in the nucleotide sequence of a nucleic acid or as variations in the amino acid sequence of a protein. Polymorphism includes the presence of one or more variations in the nucleic acid sequence or nucleic acid features of one or more loci in a population of one or more individuals. Variation may include, but is not limited to, one or more nucleotide base changes, one or more nucleotide insertions, or one or more nucleotide deletions. Polymorphism can arise from random processes in nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variations, and during the process of meiosis (e.g., unequal crossovers, genome duplication, and chromosome breaks and fusions). Variation may be commonly found or present at low frequency in a population, the former having greater utility in general plant breeding, while the latter may be associated with rare but important phenotypic variations. Useful polymorphisms include single nucleotide polymorphisms (SNPs), insertions or deletions within DNA sequences (Indels), simple sequence repeats (SSRs) of DNA sequences, restriction fragment length polymorphisms (RFLPs), and tagged SNPs. Gene markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, the 5' untranslated region of genes, the 3' untranslated region of genes, microRNAs, siRNAs, resistance loci, satellite markers, transgenes, mRNAs, dsmRNAs, transcription profiles, and methylation patterns may also be polymorphisms. In addition, the presence, absence, or variation of the aforementioned copy numbers may also constitute polymorphisms.

[0161] As used herein, “SNP” or “single nucleotide polymorphism” refers to a variation in a genome sequence that occurs when a single nucleotide (A, T, C, or G) in the genome sequence is altered or variable. “SNP marker” is present when an SNP is mapped to a site on the genome.

[0162] Where used herein, the terms “marker,” “molecular marker,” or “marker locus” are used to describe a nucleic acid or amino acid sequence that is sufficiently specific to characterize a particular locus on the genome. Any detectable polymorphic trait can be used as a marker, insofar as it is differentially inherited and exhibits linkage disequilibrium with the phenotypic trait of interest. Thus, each marker is an indicator of a particular segment of DNA having a specific nucleotide sequence. Map location provides a measure of the relative position of a particular marker to one another. Where it is stated that a trait is associated with a given marker, it will be understood that the actual DNA segment whose sequence affects the trait is generally co-separated with the marker. More accurate and reliable localization of the trait can be obtained when the marker is identified on both sides of the trait. Without actually evaluating the appearance of the trait itself (actual evaluation of a trait can be difficult and time-consuming, as it requires growing plants at a stage where the trait can be expressed and / or under environmental conditions where the trait can be expressed), the presence of a trait can be detected by a relatively simple molecular test by measuring the appearance of the marker(s) in the offspring of a cross.

[0163] It is understood that any tobacco plant of this disclosure may further include additional agronomically desirable traits by, for example, transforming with a gene construct or transgene using techniques known in the art. Examples of desirable traits include, but are not limited to, herbicide resistance, pest resistance, disease resistance, high yield, high grade index value, drought tolerance, drought quality, mechanical harvestability, retention ability, leaf quality, height, plant maturation (e.g., early maturation, early to mid-maturation, mid-maturation, mid-to-late maturation, or late maturation), stem size (e.g., small, medium, or large stem), or number of leaves per plant (e.g., few (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or many (16-21 leaves)), or any combination thereof. In one embodiment, the disclosed low-nicotine or nicotine-free tobacco plant or seeds include one or more transgenes expressing one or more insecticidal proteins, e.g., crystalline proteins of Bacillus thuringiensis, or plant insecticidal proteins derived from Bacillus cereus, such as VIP3 (see, for example, Estruch et al. (1997) Nat. Biotechnol. 15:137). In another embodiment, the tobacco plant further includes a transgenetic trait that confers resistance to brown stem rot (U.S. Patent No. 5,689,035) or resistance to cyst nematode (U.S. Patent No. 5,491,081).

[0164] The disclosure also provides PMT mutant tobacco plants having yields equivalent to those of the corresponding original tobacco plant without altered nicotine levels or total alkaloid levels. In one embodiment, the PMT mutant provides yields selected from the group consisting of approximately 1200–3500, 1300–3400, 1400–3300, 1500–3200, 1600–3100, 1700–3000, 1800–2900, 1900–2800, 2000–2700, 2100–2600, 2200–2500, and 2300–2400 pounds / acre. In another embodiment, the PMT mutant tobacco varieties offer yields selected from a group consisting of approximately 1200-3500, 1300-3500, 1400-3500, 1500-3500, 1600-3500, 1700-3500, 1800-3500, 1900-3500, 2000-3500, 2100-3500, 2200-3500, 2300-3500, 2400-3500, 2500-3500, 2600-3500, 2700-3500, 2800-3500, 2900-3500, 3000-3500, and 3100-3500 pounds / acre. In a further embodiment, PMT mutant tobacco plants provide yields of 65%–130%, 70%–130%, 75%–130%, 80%–130%, 85%–130%, 90%–130%, 95%–130%, 100%–130%, 105%–130%, 110%–130%, 115%–130%, or 120%–130% of the yield of a control plant having essentially the same genetic background as the PMT mutant. In a further embodiment, PMT mutant tobacco plants provide yields of 70%–125%, 75%–120%, 80%–115%, 85%–110%, or 90%–100% of the yield of a control plant having essentially the same genetic background as the PMT mutant.

[0165] In one embodiment, the disclosed tobacco plant (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco variety) includes modifications that confer a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) without substantially affecting a trait selected from the group consisting of yield, maturation and senescence, susceptibility to insect damage, polyamine content after flowering, chlorophyll levels, number of mesophyll cells per unit leaf area, and quality of the final product after drying.

[0166] In one embodiment, the disclosed tobacco plant comprises modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloid), further comprising a trait substantially equivalent to that of an unmodified control plant, the trait being selected from the group consisting of yield, maturation and senescence, susceptibility to insect damage, polyamine content after flowering, chlorophyll levels, number of mesophyll cells per unit leaf area, and quality of the final product after drying.

[0167] In one embodiment, the disclosed tobacco plant includes modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further includes a yield of more than 80%, more than 85%, more than 90%, more than 95%, more than 100%, more than 105%, more than 110%, more than 115%, more than 120%, more than 125%, more than 130%, more than 135%, or more than 140% of the yield of an unmodified control plant. In one embodiment, the disclosed tobacco plant includes modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further includes a yield of 70% to 140%, 75% to 135%, 80% to 130%, 85% to 125%, 90% to 120%, 95% to 115%, or 100% to 110% of the yield of an unmodified control plant. In one embodiment, the disclosed tobacco plant comprises modifications that confer a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprises yields of 70%-80%, 75%-85%, 80%-90%, 85%-95%, 90%-100%, 95%-105%, 105%-115%, 110%-120%, 115%-125%, 120%-130%, 125%-135%, or 130%-140% relative to the yield of an unmodified control plant.

[0168] In one embodiment, the disclosed low-nicotine or nicotine-free tobacco varieties are adapted for mechanical harvesting. In another embodiment, the disclosed low-nicotine or nicotine-free tobacco varieties are mechanically harvested.

[0169] In one embodiment, the tobacco plant provided is a hybrid plant. The hybrid can be produced by preventing self-pollination of the female parent plant (e.g., seed parent) of a first variety, fertilizing the female parent plant with pollen from the male parent plant of a second variety, and forming F1 hybrid seeds in the female plant. Self-pollination of the female plant can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation in the female parent plant can be prevented using a form of male sterility. For example, male sterility can be produced by male sterility (MS) or transgenic male sterility, in which the introduced gene inhibits microspore formation and / or pollen formation, or self-incompatibility. Female parent plants containing MS are particularly useful. In one embodiment, where the female parent plant is MS, pollen is collected from a male fertile plant and manually applied to the stigma of the MS female parent plant, and the resulting F1 seeds are collected.

[0170] Using plants, single-cross tobacco F1 hybrids can be formed. F1 seeds are formed by manually transferring pollen from the male parent plant to a emasculated female parent plant or a male-sterile female parent plant. Alternatively, a three-way cross can be performed, using a single-cross F1 hybrid as the female parent and crossing it with a different male parent. Another alternative is to create a double-cross hybrid by crossing the F1 offspring of two different single crosses. When forming a double-cross hybrid, using self-incompatibility is particularly advantageous, as it prevents self-pollination of the female parent.

[0171] In one embodiment, low-nicotine or nicotine-free tobacco varieties are male-sterile. In another embodiment, low-nicotine or nicotine-free tobacco varieties are cytoplasmically male-sterile. Male-sterile tobacco plants can be produced by any method known in the art. A method for producing male-sterile tobacco is described in Wernsman, EA, and Rufty, RC1987 Chapter Seventeen. Tobacco. Pages 669-698 Cultivar Development. Crop Species. WHFehr (ed.), MacMillan Publishing Go., Inc., New York, NY 761 pp.

[0172] In one embodiment, the disclosure provides a male-sterile tobacco plant, variety, or strain containing one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E.

[0173] In another embodiment, the Disclosure provides male-sterile tobacco plants, varieties, or strains derived from any tobacco plant, variety, or strain provided in any one of Tables 4A to 4E, Table 10, or Table 14.

[0174] In one embodiment, the Disclosure provides a male sterile strain dCS11. In another embodiment, the Disclosure provides a male sterile strain dCS12. In another embodiment, the Disclosure provides a male sterile strain dCS13. In another embodiment, the Disclosure provides a male sterile strain dCS14. In another embodiment, the Disclosure provides a male sterile strain dCS15. In another embodiment, the Disclosure provides a male sterile strain dCS16. In another embodiment, the Disclosure provides a male sterile strain dCS17. In another embodiment, the Disclosure provides a male sterile strain dCS18. In another embodiment, the Disclosure provides a male sterile strain dS697.

[0175] In further embodiments, the tobacco parts provided include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, sheaths, embryos, endosperm, explants, callus, tissue cultures, shoots, cells, and protoplasts. In one embodiment, the tobacco parts provided do not include seeds. In one embodiment, the disclosure provides organs that are not cells, tissues, and germline material of the tobacco plant and do not mediate the natural reproduction of the plant. In another embodiment, the disclosure also provides organs that are cells, tissues, and germline material of the tobacco plant and mediate the natural reproduction of the plant. In yet another embodiment, the disclosure provides cells, tissues, and organs of the tobacco plant that are unable to sustain themselves through photosynthesis. In yet another embodiment, the disclosure provides somatic tobacco plant cells. Somatic cells, unlike germline cells, do not mediate plant reproduction.

[0176] Cells, tissues, and organs may originate from seeds, fruits, leaves, cotyledons, hypocotyls, growing points, embryos, endosperm, roots, shoots, stems, sheaths, flowers, inflorescences, stalks, pedicels, styles, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarps, phloem, and vascular tissues. In another embodiment, the disclosure provides chloroplasts of a tobacco plant. In yet another embodiment, the disclosure provides epidermal cells, stomatal cells, leaves or root hairs, storage roots, or tubers. In yet another embodiment, the disclosure provides tobacco protoplasts.

[0177] Those skilled in the art understand that tobacco plants reproduce naturally through seeds, rather than through asexual or vegetative propagation. In one embodiment, the disclosure provides tobacco endosperm. In another embodiment, the disclosure provides tobacco endosperm cells. In a further embodiment, the disclosure provides male or female sterile tobacco plants that cannot reproduce without human intervention.

[0178] In one embodiment, the disclosure provides nucleic acid molecules having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with sequences and fragments selected from the group consisting of SEQ ID NOs: 1 to 10. In one embodiment, the disclosure provides polypeptides or proteins having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with amino acid sequences selected from the group consisting of SEQ ID NOs: 11 to 15.

[0179] As used herein, the terms “sequence identity” or “identity” in the context of two polynucleotide or polypeptide sequences refer to identical residues in the two sequences when aligned for the greatest correspondence across a specified comparison window. When a percentage of sequence identity is used in relation to proteins, it is recognized that non-identical residue positions are often differed by conservative amino acid substitutions, where the amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. If sequences differ by conservative substitutions, the percentage of sequence identity may be adjusted upward to compensate for the conservative nature of the substitutions.

[0180] This disclosure further provides a method for producing a tobacco product containing tobacco material from the disclosed tobacco plant. In one embodiment, the method includes adjusting aged tobacco material made from the tobacco plant to increase its moisture content from about 12.5% ​​to about 13.5% to about 21%, and blending the adjusted tobacco material to produce a desired blend. In one embodiment, the method for producing a tobacco product further includes casing or flavoring the blend. Generally, during the casing process, casing or source materials are added to the blend to balance the chemical composition, thereby enhancing their quality and developing specific desired flavor characteristics. Further details of the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.

[0181] Furthermore, the tobacco material provided may be processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzymatic treatment, expansion, and / or drying. Both fermented and unfermented tobaccos may be processed using these techniques. Suitable examples of processed tobaccos include dark air drying, dark flame drying, burley, hot air drying, and products of filler or wrapper, as well as whole leaf stemming operations. In one embodiment, the tobacco fiber contains up to 70% dark tobacco by fresh weight. For example, tobacco may be modified by heating, sweetening, and / or pasteurization steps, as described in U.S. Publication No. 2004 / 0118422 or U.S. Publication No. 2005 / 0178398.

[0182] The provided tobacco material can be fermented. Fermentation is typically characterized by an initial high moisture content, heat generation, and a 10-20% decrease in dry weight. See, for example, U.S. Patents 4,528,993, 4,660,577, 4,848,373, and 5,372,149. Fermentation can alter the aroma of the leaves and change either or both of the color and texture of the leaves. Additionally, during the fermentation process, release gases can be produced, oxygen can be taken in, the pH can be altered, and the amount of moisture retained can be changed. See, for example, U.S. Publication No. 2005 / 0178398 and Chapter 1 of Tso (1999, Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Dried or dried and fermented tobacco can be further processed (e.g., cut, expand, blend, mill, or grind) before being incorporated into oral products. The tobacco is, in some cases, long-cut, fermented, dried, moist tobacco with an oven volatile content of 48-50% by weight before being mixed with copolymers and optionally with flavorings and other additives.

[0183] In one embodiment, the tobacco material provided can be processed to a desired size. In one embodiment, tobacco fibers can be processed to have an average fiber size of less than 200 micrometers. In one embodiment, the tobacco fibers are 75 to 125 micrometers. In another embodiment, the tobacco fibers are processed to have a size of 75 micrometers or less. In one embodiment, the tobacco fibers include long-cut tobacco that can be cut or shredded to a width of about 10 cuts / inch to about 110 cuts / inch and a length of about 0.1 inches to about 1 inch. Double-cut tobacco fibers can have varying particle sizes such that about 70% of the double-cut tobacco fibers have a mesh size of -20 mesh to 80 mesh.

[0184] The tobacco material provided may be processed to have a total oven volatile content of about 10% by weight or more, about 20% by weight or more, about 40% by weight or more, about 15% to about 25% by weight, about 20% to about 30% by weight, about 30% to about 50% by weight, about 45% to about 65% by weight, or about 50% to about 60% by weight. Those skilled in the art will understand that “moist” tobacco typically refers to tobacco having an oven volatile content of about 40% to about 60% by weight (e.g., about 45% to about 55% by weight, or about 50% by weight). As used herein, “oven volatile” is determined by drying the sample in a preheated forced-fume oven at 110°C for 3.25 hours and then calculating the percentage of weight loss of the sample. Oral products may have a total oven volatile content different from the oven volatile content of the tobacco fibers used to make the oral products. The processing steps described can reduce or increase the oven volatile content.

[0185] While the disclosure has been described in general terms, the same can be more readily understood by referring to the following examples, which are provided as illustrations and are not intended to limit the disclosure, unless otherwise specified. [Examples]

[0186] Example 1: Expression profiling of 5 types of PMT genes Nicotine biosynthesis is initiated by the conversion of the polyamine putrescine to N-methylputrescine by the enzyme putrescine N-methyltransferase (PMT). This is the step in which the precursor metabolite is involved in nicotine biosynthesis. The genes encoding PMT (PMT1a, PMT1b, PMT2, PMT3, and PMT4) are located in the tobacco (nicotiana tabacum) genome. Table 1A lists the genomic DNA sequences, cDNA sequences, and protein sequences of the five PMT genes. Tables 1B and 1C provide sequence identity between the five PMT genes. Pooled expression levels from pre-pruning to harvest provide support for PMT1a and PMT3 representing the two major PMT genes, without being limited to any particular theory (Figure 1).

[0187] (Table 1A) Sequences of the PMT genes of five tobacco species TIFF2026083235000002.tif51128

[0188] (Table 1B) cDNA sequence identity between five tobacco PMT genes determined by Clustal2.1 TIFF2026083235000003.tif34128

[0189] (Table 1C) Protein sequence identity between five tobacco PMT genes determined by Clustal2.1 TIFF2026083235000004.tif34128

[0190] (Table 1D) PMT1b genome sequence (SEQ ID NO: 1) Annotation TIFF2026083235000005.tif80128

[0191] (Table 1E) PMT1b genome sequence (SEQ ID NO: 2) with annotations TIFF2026083235000006.tif80128

[0192] (Table 1F) PMT2 genome sequence (SEQ ID NO: 3) Annotation TIFF2026083235000007.tif80128

[0193] (Table 1G) PMT3 genome sequence (SEQ ID NO: 4) with annotations TIFF2026083235000008.tif80128

[0194] (Table 1H) PMT4 genome sequence (SEQ ID NO: 5) Annotation TIFF2026083235000009.tif80128

[0195] Example 2: PMT genome editing and development of tobacco strains PMT knockout mutants are generated by editing various PMT genes. Tobacco protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding either the Genome Editing Technology 1 (GET1) protein or the Genome Editing Technology 2 (GET2) protein, and specific guide RNAs (gRNAs) that target PMT genes at desired locations. Table 2 lists the gRNA sequences used for PMT editing. Several gRNAs (e.g., numbers 6 and 7) are pooled together to target multiple PMT genes in a single transfection.

[0196] Next, the transfected protoplasts are immobilized on 1% agarose beads and subjected to tissue culture. When the callus grows to approximately 1 mm in diameter, it is seeded onto a TOM2 plate. The callus is screened for insertions or deletions (indels) at target sites using fragment analysis. Candidates showing size shifts compared to wild-type controls are selected for further culture, and the resulting shoots are tested again by fragment analysis to confirm the presence of indels. Rooted shoots are potted and sequenced at target sites to determine the exact deleted sequence. Young leaves are collected from each plant and PCR amplified for PMT fragments using phirekit. A PMT library is indexed for each strain, 384 strains are pooled, and sequenced using Miseq.

[0197] SNP analysis is performed to determine both the exact sequence of the edited pmt mutant allele and the conjugation status at each PMT locus. Table 3 provides conjugation information for representative edited plants. Tables 4A to 4E provide indel sequence information for each edited strain of various tobacco varieties (e.g., K326, TN90, NLM, Oriental). Tables 5A to 5E provide the genome sequence of approximately 40 nucleotides from each pmt mutant allele with an edit site in the middle of the genome sequence (e.g., 20 nucleotides on each side of the deletion or insertion site).

[0198] (Table 2) gRNA sequences used in two genome editing technologies and their target genes. "Y" indicates that the gRNA targets its PMT gene, while "-" indicates that the gRNA does not target its PMT gene. TIFF2026083235000010.tif94156

[0199] (Table 3) Conjugation of individual PMT loci in selected PMT mutants in various backgrounds generated by genome editing using GET2. Number 1 indicates homozygosity for a single mutant allele. Numbers 2-5 represent heterozygous allele combinations with 2-5 indels. A hyphen indicates no data. Detailed genotyping information is shown in Tables 4A-4D. TIFF2026083235000011.tif187136TIFF2026083235000012.tif226136TIFF2026083235000013.tif226136TIFF2026083235000014.tif61136

[0200] (Table 4A) Mutant PMT alleles at K326 generated by genome editing using GET2. The location of each editing site (e.g., indel) is relative to the nucleotide number on the corresponding cDNA sequence of each PMT gene. For example, strain 17GH1678 has both allele mutations in PMT1b. One of the two alleles has a deletion of four nucleotides corresponding to nucleotides 416-419 of the PMT1b cDNA sequence. The other allele has a deletion of two nucleotides corresponding to nucleotides 418-419 of the PMT1b cDNA sequence. Sequence numbers are assigned and shown for sequences exceeding 10 nucleotides. TIFF2026083235000015.tif217113TIFF2026083235000016.tif217153TIFF2026083235000017.tif21765

[0201] (Table 4B) TN90 mutant pmt allele generated by genome editing using GET2 TIFF2026083235000018.tif233141TIFF2026083235000019.tif233150TIFF2026083235 000020.tif233149TIFF2026083235000021.tif233153TIFF2026083235000022.tif23369

[0202] (Table 4C) NLMz mutant pmt allele generated by genome editing using GET2. NLMz refers to a narrow-leaf Mador variety containing three loss-of-function mutations in three nicotine demethylase genes (CYP82E4, CYP82E5v2, and CYP82E10). TIFF2026083235000023.tif230133TIFF2026083235000024.tif230150TIFF2026083235000025.tif230154TIFF2026083235000026.tif23083

[0203] (Table 4D) PMT allele of Oriental tobacco mutant generated by genome editing using GET2 TIFF2026083235000027.tif219140TIFF2026083235000028.tif219152TIFF2026083235000029.tif21959

[0204] (Table 4E) PMT allele of NLM(PhPh) tobacco mutant generated by genome editing using GET1 TIFF2026083235000030.tif23632

[0205] (Table 5A) A list of exemplary mutant alleles obtained from the PMT1b gene. The sequences of the mutant alleles listed herein and in Tables 5B to 5E represent approximately 40-nucleotide-length genomic sequences from each edited PMT gene with an editing site in the middle of the genomic sequence (e.g., 20 nucleotides on each side of the deletion or insertion site). These mutant alleles correspond to those listed in Tables 4A to 4E. TIFF2026083235000031.tif196119TIFF2026083235000032.tif196145TIFF2026083235000033.tif196154TIFF2026083235000034.tif19635

[0206] (Table 5B) List of exemplary mutant alleles obtained from the PMT1a gene TIFF2026083235000035.tif188145TIFF2026083235000036.tif188154TIFF2026083235000037.tif188137

[0207] (Table 5C) List of exemplary mutant alleles obtained from the PMT2 gene TIFF2026083235000038.tif173143TIFF2026083235000039.tif173154TIFF2026083235000040.tif173154

[0208] (Table 5D) List of exemplary mutant alleles obtained from the PMT3 gene TIFF2026083235000041.tif196145TIFF2026083235000042.tif196154TIFF2026083235000043.tif196119

[0209] (Table 5E) List of exemplary mutant alleles obtained from the PMT4 gene TIFF2026083235000044.tif202145TIFF2026083235000045.tif202145

[0210] Example 3: Alkaloid analysis of PMT-edited strains Genome-edited tobacco plants, along with a control, were grown in a greenhouse in 10-inch pots using 75 PPM fertilizer. At the flowering stage, the plants were pruned, and two weeks after pruning, leaf blade samples were collected from the third, fourth, and fifth leaves from the top, and alkaloid levels were measured (see Tables 6A–6C). This measurement followed the method outlined in CORESTA Method 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography (February 2005), and the method defined in the Centers for Disease Control and Prevention's Protocol for the Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco Products, published in Federal Register Vol. 64, No. 55, March 23, 1999 (revised Vol. 74, No. 4, January 7, 2009).

[0211] In short, approximately 0.5 g of tobacco is extracted using liquid / liquid extraction into an organic solvent containing an internal standard, and analyzed by gas chromatography (GC) with flame ionization detection (FID). The results can be reported as weight percentages (W%), either as is or on a dry weight basis. Oven volatile matter (OV) determination is required to report data on a dry weight basis. Unless otherwise specified, the total alkaloid levels, individual alkaloid levels, or nicotine levels shown herein are on a dry weight basis (e.g., percentage of total alkaloids or percentage of nicotine).

[0212] The plants are also planted in fields, harvested, and tested for alkaloid and TSNA levels in dried tobacco. Both leaf yield and leaf grade are also evaluated for PMT-edited plants. Furthermore, different mutant combinations of individual PMT genes are generated and tested (e.g., single, double, triple, or quadruple mutants).

[0213] Example 4: Comparison of the quintuple PMT knockout mutant with other low-alkaloid tobacco plants. The quintuple PMT knockout mutant CS15 (NLM (Ph Ph) background; see Table 4E for genotype) was grown alongside a PMT RNAi transgene strain (VA359 background, described in U.S. Patent No. 2015 / 0322451) and a low-nicotine KY171 ("LN KY171") variety (KY171 background with double mutations in nic1 and nic2). Leaves were harvested and dried via dark-fire drying. Each strain was analyzed for nicotine levels, total alkaloid levels, leaf yield, and leaf quality (Figures 2-5). The data showed that suppressing PMT gene activity by editing all five PMT genes reduced nicotine levels without affecting leaf yield or leaf quality.

[0214] Example 5: Obtain a tobacco strain having edited mutant alleles in one or more PMT genes. Tobacco strains with mutations in individual PMT genes or selected combinations of PMT genes are obtained from the tobacco strains listed in Table 3. Cross quintuple, quadruple, triple, or double mutants (having mutations in 5, 4, 3, or 2 PMT genes, respectively) with non-mutant control plants and select segregated offspring plants for specific PMT mutation combinations. Tables 7A-7E represent possible mutant combinations. Each mutant gene can be either homozygous or heterozygous for the mutation. Single, double, triple, quintuple, or quadruple mutants can be generated using each of the mutant alleles listed in Tables 4A-4E and Table 10. Exemplary individual PMT mutant alleles are listed in Tables 9A-9E.

[0215] Example 6: Further reduction of total alkaloids by combining the pmt mutation with mutations in other genes. To further reduce total alkaloids and / or selected individual alkaloids, pmt mutants are combined with mutations in additional genes involved in tobacco alkaloid biosynthesis, such as quinolate phosphoribosyltransferase (QPT) or quinolate synthase (QS). Briefly, gene editing is used to mutate selected QPT and / or QS genes in the desired pmt mutant background (e.g., quadruple or quintuple pmt mutants). Alkaloid levels and TSNA levels are tested in dried tobacco in the resulting qpt / pmt or qs / pmt mutants. Both leaf yield and leaf grade are also evaluated.

[0216] (Table 6A) Alkaloid levels in PMT-edited strains of K326 (as weight percentage per gram of leaf blade (dry weight), shown here, in Tables 6B, 6C, and 7) TIFF2026083235000046.tif104128

[0217] (Table 6B) Alkaloid levels in TN90 PMT edited strains TIFF2026083235000047.tif188128

[0218] (Table 6C) Alkaloid levels in PMT-edited strains of Narrow Leaf Madol (NLM) TIFF2026083235000048.tif131128

[0219] (Table 7) Relative changes in nicotine and total alkaloid levels in quintuple PMT knockout mutants across various varieties. The mean percentage levels of nicotine and total alkaloids are calculated based on percentage level data from individual strains, as shown in Tables 6A–6C. The relative changes reflect the nicotine or total alkaloid levels in the quintuple PMT mutants compared to their controls. TIFF2026083235000049.tif83128

[0220] (Table 8A) List of mutants obtained from various genotype combinations of the five PMT genes: single-gene mutations TIFF2026083235000050.tif33128

[0221] (Table 8B) List of mutants obtained from various genotype combinations of the five PMT genes: double gene mutations TIFF2026083235000051.tif59128

[0222] (Table 8C) List of mutants obtained from various genotype combinations of the five PMT genes: Triple gene combinations TIFF2026083235000052.tif59128

[0223] (Table 8D) List of mutants obtained from various genotype combinations of the five PMT genes: Quadruple gene combinations TIFF2026083235000053.tif33128

[0224] (Table 8E) List of mutants obtained from various genotype combinations of the five PMT genes: Quintuple gene combinations TIFF2026083235000054.tif12128

[0225] Example 7: PMT genome editing and development of tobacco strains Further PMT knockout mutants are generated by editing all five PMT genes (PMT1a, PMT1b, PMT2, PMT3, and PMT4) in different tobacco strains. Tobacco protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding genome editing technology (GET2) proteins and specific guide RNAs (gRNAs) that target PMT genes at desired locations. Table 9 lists the gRNA sequences used for PMT editing. Several gRNAs (e.g., numbers 6 and 7) are pooled together to target multiple PMT genes in a single transfection.

[0226] (Table 9) Guide RNA for GET2 used in Example 7. "Y" indicates that the gRNA can target its PMT gene, while "-" indicates that the gRNA does not target its PMT gene. TIFF2026083235000055.tif49161

[0227] Next, the transfected protoplasts are immobilized on 1% agarose beads and subjected to tissue culture. When the callus grows to approximately 1 mm in diameter, it is seeded onto a TOM2 plate. The callus is screened for insertions or deletions (indels) at target sites using fragment analysis. Candidates showing size shifts compared to wild-type controls are selected for further culture, and the resulting shoots are tested again by fragment analysis to confirm the presence of indels. Rooted shoots are potted and sequenced at target sites to determine the exact deleted sequence. Young leaves are collected from each plant and PCR amplified for PMT fragments using phirekit. A PMT library is indexed for each strain, 384 strains are pooled, and sequenced using Miseq.

[0228] SNP analysis is performed to determine both the exact sequence of the edited PMT mutant allele and the conjugation status at each PMT locus. Table 10 provides indel sequence information for each edited strain of various tobacco varieties (e.g., Basma, K326, Caterini, TN90, Izmir).

[0229] (Table 10) Various strain mutant PMT alleles generated by genome editing using GET2. The location of each editing site (e.g., indel) corresponds to a nucleotide number on the corresponding cDNA sequence of each PMT gene (e.g., SEQ ID NO: 6 for PMT1a, SEQ ID NO: 7 for PMT1b, SEQ ID NO: 8 for PMT2, SEQ ID NO: 9 for PMT3, SEQ ID NO: 10 for PMT4). Sequence numbers are assigned and shown for sequences with more than 10 nucleotides. TIFF2026083235000056.tif181128TIFF2026083235000057.tif181130TIFF2026083235000058.tif181128

[0230] Table 11 provides the chain length (in nucleotide units) of each PMT indel of each gene within each strain, as provided in Table 10.

[0231] (Table 11) Chain length (in nucleotide units) of each indel in the selected strains provided in Table 10 TIFF2026083235000059.tif186146

[0232] Tables 12A to 12E provide the genome sequences of approximately 90 nucleotides from each PMT mutant allele with an editing site in the center of the genome sequence (e.g., 45 nucleotides on each side of the deletion or insertion site).

[0233] (Table 12A) A list of exemplary mutant alleles obtained from the PMT1a gene. The sequences of the mutant alleles listed herein represent approximately 90-nucleotide lengths of genomic sequences from each edited PMT1a gene with an edit site in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. Lowercase letters in the reference allele sequence (SEQ ID NO: 6) indicate the nucleotides deleted in the mutant allele. TIFF2026083235000060.tif25588TIFF2026083235000061.tif255115TIFF20260832350 00062.tif255119TIFF2026083235000063.tif255119TIFF2026083235000064.tif255100

[0234] (Table 12B) A list of exemplary mutant alleles obtained from the PMT1b gene. The sequences of the mutant alleles listed herein represent approximately 90-nucleotide lengths of genomic sequences from each edited PMT1b gene with an edit site in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. Lowercase letters in the reference allele sequence (SEQ ID NO: 7) indicate the nucleotides deleted in the mutant allele. TIFF2026083235000065.tif255103TIFF2026083235000066.tif255119TIFF2026083235 000067.tif255119TIFF2026083235000068.tif255119TIFF2026083235000069.tif25588

[0235] (Table 12C) List of exemplary mutant alleles obtained from the PMT2 gene. The sequences of the mutant alleles listed herein represent approximately 90-nucleotide lengths of genomic sequences from each edited PMT2 gene with an editing site in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. Lowercase letters in the reference allele sequence (SEQ ID NO: 8) indicate the nucleotides deleted in the mutant allele. TIFF2026083235000070.tif255104TIFF2026083235000071.tif255119TIFF2026083235 000072.tif255119TIFF2026083235000073.tif255119TIFF2026083235000074.tif25588

[0236] (Table 12D) A list of exemplary mutant alleles obtained from the PMT3 gene. The sequences of the mutant alleles listed herein represent approximately 90-nucleotide-long genomic sequences from each edited PMT3 gene with an editing site in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. Lowercase letters in the reference allele sequence (SEQ ID NO: 9) indicate the nucleotides deleted in the mutant allele. TIFF2026083235000075.tif255104TIFF2026083235000076.tif255119TIFF2026083235 000077.tif255119TIFF2026083235000078.tif255119TIFF2026083235000079.tif25588

[0237] (Table 12E) A list of exemplary mutant alleles obtained from the PMT4 gene. The sequences of the mutant alleles listed herein represent approximately 90-nucleotide lengths of genomic sequences from each edited PMT4 gene with an editing site in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted sequence site). The mutant alleles correspond to the indels provided for each strain in Table 10. Lowercase letters in the reference allele sequence (SEQ ID NO: 10) indicate the nucleotides deleted in the mutant allele. TIFF2026083235000080.tif255104TIFF2026083235000081.tif255119TIFF2026083235 000082.tif255119TIFF2026083235000083.tif255119TIFF2026083235000084.tif25588

[0238] Example 8. Alkaloid analysis of PMT-edited strains Homozygous genome-edited tobacco strains from Example 7, as well as control strains, were grown in the field. At the flowering stage, the plants were thinned, and two weeks after thinning, leaf blade samples were collected from the third, fourth, and fifth leaves from the top of the plant, and alkaloid levels were measured (see Tables 13A-13C). This measurement was performed using the method according to CORESTA Method 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography (February 2005), and the method defined in the Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products of the Centers for Disease Control and Prevention, published in Federal Register Vol. 64, No. 55, March 23, 1999 (Revised Vol. 74, No. 4, January 7, 2009).

[0239] Approximately 0.5 g of tobacco is extracted using liquid / liquid extraction into an organic solvent containing an internal standard, and analyzed by gas chromatography (GC) with flame ionization detection (FID). Results can be reported as weight percentages (W%), either directly or on a dry weight basis. Oven volatile matter (OV) determination is required to report data on a dry weight basis. Unless otherwise specified, the total alkaloid levels, individual alkaloid levels, or nicotine levels shown herein are on a dry weight basis (e.g., percentage of total alkaloids or percentage of nicotine).

[0240] The plants are also planted in fields, harvested, and tested for alkaloid and TSNA levels in dried tobacco. Both leaf yield and leaf grade are also evaluated for PMT-edited plants.

[0241] (Table 13A) Nicotine analysis of K326 and TN90 PMT edited strains two weeks after flowering. TIFF2026083235000085.tif20873

[0242] (Table 13B) Nicotine analysis of K326 and TN90 PMT edited strains two weeks after flower thinning. TIFF2026083235000086.tif21270

[0243] (Table 13C) Nicotine analysis of Caterini and Basma PMT edited strains two weeks after flowering. TIFF2026083235000087.tif118128

[0244] Example 9. Development of male-sterile PMT-edited strains PMT-edited hybrid strains are developed using strains derived from Example 7. These hybrid strains are grown in the field and used as precursors for male-sterile strains. See Table 14.

[0245] (Table 14) PMT-edited extremely low nicotine male sterile strains TIFF2026083235000088.tif91128

[0246] Example 10. The PMT-edited strain is resistant to mold during drying. Leaves of tobacco collected from several low-alkaloid tobacco strains are subjected to a standard air-drying method. After completion of drying, the tobacco leaves are examined for mold.

[0247] Tobacco from LA BU21 shows more mold infection than TN90 LC, the TN90 variety containing an RNAi construct that downregulates all five PMT genes, the TN90 variety containing an RNAi construct that downregulates the alkaloid biosynthesis gene PR50, and four PMT-edited strains (CS47, CS59, CS63, and CS64) within the TN90 genetic background. See Table 15 and Figures 6A - 6E and 7.

[0248] (Table 15) Mold damage per tobacco strain. "G" indicates little / no mold, "S" indicates some mold, and "B" indicates significant mold. The percentage of mold refers to the proportion of air-dried tobacco sticks in each category of mold damage. TIFF2026083235000089.tif23049

[0249] Sequence information SEQUENCE LISTING <110> ALTRIA CLIENT SERVICES LLC <120> COMPOSITIONS AND METHODS BASED ON PMT ENGINEERING FOR PRODUCING TOBACCO PLANTS AND PRODUCTS HAVING ALTERED ALKALOID LEVELS <150> US 62 / 703,775 <151> 2018-07-26 <150> US 62 / 848,159 <151> 2019-05-15 <16C> 729 <170> PatentIn version 3.5 <210> 1 <211> 4317 <212> DNA <213> Nicotiana tabacum <400> 1 agtctcagac ttaatccagt atatcccatc ttatctcaca ttatcccatc aaatgtgaga 60 ttattttatc tcatctctca tgtggtataa attagtcatg aaattataat ctcgggataa 120 tttagtccgc gtaccaaacg accccaagtg ctttattgtt ttcttattga cagagtaagt 180 gtatgggaac ttaccataga aatccttcgc tcaaaaggaa atacttgcaa gaactggcca 240 aacccaaaga tgaataaact caattacttg attcttaaac tcttaaaaat gaattcaatg 300 gagaaggaaa atatttccag tgtaaacaca agtgaatgaa gagaagccaa aataatctct 360 atcattcaag ccttaggtgg agattaaaaa aattatttac tttcttatca aagtaatagg 420 tgatcaacag ctttcgtaaa acgtcattag gagaatatta taatctcttt tatgctgaag 480 aacccacata aggaagatca taaaatacat gactttcaga tgacttcttg gagctttatt 540 tttaaagagt ggctagctgg tcagcaaaga ggtgctcgtc agatatcata aaattttact 600 attatttgtt ttaagaggga gatggggcac acatgcttgt vakaaaagta agaggagaa 660 aggaacaga agaggaaata gatttggggg gggggggggg gttcacaat chaaaaaat 720 ttttaaaatg gagagaaaa tgagcacaca catatactaa caaaattta ctaataattg 780 caccgagaca aacttatatt ttagttccaa atgtcagtc taaccctgca cgttgtaatg 840 aatttttaac tattatta tatcgagttg cgccctccac tcctcggtgt ccaaattgta 900 tttaaatgca tagatgttta ttgggagtgt acagcaagct ttcggaaat acaaccata 960 atactttctc ttctcaatt tgtttagttt aattttgaa atggagtca tatctaccaa 1020 cacaaatggc tctaccatct tcaagaatgg tgccattccc atgaacggcc accaaatgg 1080 cactctgaa cacctcaacg gctaccagaa tgcacttcc aaacaccaaa acgggcacca 1140 gatggcact ttcgaacatc ggaacggcca ccagaatggg acatccgaac aacagaacgg 1200 gatacagc catgacaatg gcaacgagct actgggaagc tccgactcta ttaagcctgg 1260 ctggttttca gagtttagcg cattatggcc aggttagtac tagaaagca actcaatgc 1320 atcggcctct tgttgctact aaatatagag agctatcata cttttaggga ctaactaaaa 1380 aggaaagatt atcacaggga cgaagtgagc agttaacttc gcatattatc agacgcatta 1440 atttgaaata atcgaatttt gcaggtgaag cattctcact taaggttgag aagttactat 1500 tccaggggaa gtctgattac caagatgtca tgctctttga ggtaattaat attctaatac 1560 acatgcttta atttaaagtg atacttttaa tttactttta gtttattgca tgtgcacgta 1620 cagtcagcaa cttatgggaa ggttctgact ttggatggag caattcaaca tacagagaat 1680 ggtggatttc catacactga aatgattgtt catctaccac ttggttccat cccaaaccca 1740 aaaaaggttt tgatcatcgg cggaggaatt ggttttacat tattcgaaat gcttcgttat 1800 ccttcaatcg aaaaaattga cattgttgag atcgatgacg tggtagttga tgtaagtcaa 1860 acttcttttta cccacataaaa gaaaatgatt tagattgcaa ttctttttat tttcttaaaa 1920 gaataaatat attctctttt tttttttaaa acaaaattct ctttcttaca ggtatccaga 1980 aaatttttcc cttatctggc agctaatttt aacgatcctc gtgtaaccct agttctcgga 2040 gatggtgcgt atatgatagt ctcgttttat attttatttc acttgatttt tacctttttt 2100 tgtggttaat taatcatcta ccattggttc tctttacctt caggagctgc atttgtaaag 2160 gctgcacaag cgggatatta tgatgctatt atagtggact cttctgatcc cattggtacg 2220 ctattactat ttaataccaa gactattctt attaaataag ctactaagaa actaattgaa 2280 taattaataa acgtaactgt aattgatttc taaaataata tatataattt caggtccagc 2340 aaaagatttg tttgagaggc cattctttga ggcagtagcc aaagccctta ggccaggagg 2400 agttgtatgc acacaggctg aaagcatttg gcttcatatg catattatta agcaaatcat 2460 tgctaactgt cgtcaagtct ttaagggttc tgtcaactat gcttggacaa ccgttccaac 2520 atatcccacg tattcttttt ctctctct cttcctgtct tttcgatgc aatgtaaatt 2580 tataaaattg gaagtccgtt ttactttct atagacgtag atcctaaaat tgtcaagaaa 2640 tggagaattg acttacaaga aaaatcaact tctttcatt tactattcttt tttggtgaca 2700 aactttactt attatttcgt tctaaaatga aaatttattt ttatatttta aaatattta 2760 gctttaaact tttaatttta cttgttatat ttttaataa aaagatttat agtcaaataa atgttgtgac catataaaaa cctccgcatt tttaagatca tagtttcag agtcaaacga gttaatttat ttttagtatg ccggtgcgga gtcaaattat gtcataaaaa ttgaaacgga gtgagaacat ttttatttcg agtaaacttt caaggtattg tgtttaattt caagtgatac tgatcaatga tgtcttaaat attttgattt cagcggtgtg atcggtata tgctctgctc tactgaaggg ccagaagttg acttcaaga tccagtaaat ccaattgaca aagagacaac tcaagtcaag tccaaattag gacctctcaa gttctacaac tctgatgtaa cttcatatct cacaatttct ttttccgttt tactgtatgt tcttcgtcaa attttataac taactctttt catattgtct tttttttcag attcacaaag cagcattcat tttaccatct ttcgccagaa gtatgatcga gtcttaatca agtgaatca gaacactggt agtacaatca ttggaccaag atcgagtctt aatcaagtga father gtattgtagg agaattctgc agtaattatc ataatttcca attcacaatc attgtaaaat tctttctctg tggtgtttcg tactttaata taaatttcc tgctgaagtt ttgaatcgac gttcaactc aatcctcgca 3540 aatcagttca taccctt tcagtgtact atagtaaca aatctcatag taccgtggt 3600 gttgttttt gactatgaga tttcgatct tttgttctt gttttggaat 3660 tgtattgaat atatttgtaa cttactatgt atctaaatcg gctagtctct cttttttaat 3720 aaatggcgtc attgccgttg tagtttgttt actcggtact attttatcag cagtttcgat 3780 ttgttttgctc atttttaga agagggttga aatgtctttg ttgtaagata actttaccat 3840 atcttataga ttagtataga atgacccgtt ctgagccggg gccaaactta tattatttta 3900 taaaaagaat ggataaattt ctttgatatg tatcagttta ttgttattaa ttagagatat 3960 agtttgagaa gctcaatgcg atgaagaa gtaacatcgc tatattaac taatcacaa 4020 atagcacttt gaataagtct tcggatccca tgttgtagta ggacaaacaa taactttgtt 4080 tctatttatt tatcgttcta tttcacca tcaacaaaa agtaaatttg tttctattta 4140 gtttgaatag tcgcgtagaa aattcggtag atagcagac aaagaaaata attcaagcc 4200 aaaaatagat catatttact gttgttcttt cagcaactac tattttgcat cagttacaag 4260 accaatatgc tttaccgtat aacaaaaata taaatgtata tgccagtaaa ttacaga 4317 <210> 2 <211> 4210 <212> DNA <213> Nicotiana tabacum <400> 2 gaatttgta aatgcttttt ttggctcagt gattttgtgg agttgggaaa aatcctattg 60 gaaagtaggt cgtggtttt tcaccttttg aaccaggtat ttttcatgta aaaatacttg 120 tgttctttac ttttgcatt tactattcca caactgtagt gtaaggaaca cgtagaagaa 180 ccatgtccta taatctgtgc acgcgaaaaa ttggacacca cgcaaatcac ccttattgtg 240 tgtcattgaa atataaaaca tcaagctcaa tcatagatta atcttttttt agtaccaaca 300 tcatatgcaa aaatcaattc aacccccaaa acataataca accaatgtta atgcaatatc 360 tctgctgcta tcacgaaaat aattgctct cacgaaagta ggatacatta tgtaggttac 420 atcacataga ggtaatctaa agctcccaat aataagatgt gtaatgttga ttatgtagaa 480 atttgccagg ttattagaa taacaagaa gaggagaaa aaagtacaat ttacctgaac 540 tcttgaatgt atcctacaaa taacctagac ttcatggacg tcagttgtca gtttacttt 600 gttttaatgg tacatcattt gtcaatact tattttggt aaaaacagtt ttgcctagg 660 agtaaaaga tccggagta gaagcagac gattaagca atttttaaaa aaggagag 720 aattaatga gcacacat atactagtga aattagggta ctaatttact addaattgca 780 ccgagacaaa cttatattt agttccaaaa tgtcagtcta accctgcacg ttgtaataaa 840 ttttatactc tattatta tatcgagttg cgccctccac tcctcggtgt ccaaattgta 900 tttaaatgca tagatgttta atgggagtgt acagcaagct ttcggaaat acaaccata 960 atactttctc ttctcaatt tgtttagttt aattttgaa atggagtca tatctaccaa 1020 cacaaatggc tctaccatct tcaagaatgg tgccattccc atgaacggcc accaaatgg 1080 cactctgaa cacctcaacg gctaccagaa tgcacttcc aaacaccaaa acgggcacca 1140 gatggcact ttcgaacatc ggaacggcca ccagaatggg acatccgaac aacagaacgg 1200 gacaatcagc catgacaatg gcaacgagct actgggaagc tccgactcta ttaagcctgg 1260 ctggttttca gagtttagcg cattatggcc aggttagtat taagaaagaa actcaaatgc 1320 atcgtactct tgtattttgg tttgtgtata atttataata tggataaatt atgacgaagt 1380 aaactcgcat acattaattt gaaataatct aatttgcag gtgaagcatt ctcacttaag 1440 gttgagaagt tactattcca ggggaagtct gattaccaag atgtcatgct ctttgaggta 1500 attaatattc tgatacacat gctttaatct aaagtgatac ttttaattta cttttagttt 1560 attgcatgtg cacgtacagt cagcaactta tgggaaggtt ctgactttgg atggagcaat 1620 tcaacataca gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg 1680 ttccatccca aacccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt 1740 cgaaatgctt cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt 1800 agttgatgta agtcaaactt cttttaccca cataaagaaa atgatttaga ttgcaattct 1860 ttttatttt ctaaaagaat aatatattc tctcttttttt ttttaaaac aaaattctct 1920 ttcttacagg tatccagaaa attttccct tatctggcag ctaattttaa cgatcctcgt 1980 gtaaccctag ttctcggaga tggtgcgtat atgatagtct cgttttatat tttatttcac 2040 ttgattttta cctttttttg tggttaatta atcatctacc attggttctc tttaccttca 2100 ggagctgcat ttgtaaaggc tgcacaagcg ggatattatg atgctattat agtggactct 2160 tctgatccca ttggtacgct attactattt aataccaaga ctattcttat tatataagct 2220 actaagaaac taattgaata attaataaac gtaactgtaa ttgatttcta aaataatata 2280 tataatttca ggtccagcaa aagatttgtt tgagaggcca ttctttgagg cagtagccaa 2340 agcccttagg ccaggaggag ttgtatgcac acaggctgaa agcatttggc ttcatatgca 2400 tattattaag caaatcattg ctaactgtcg tcaagtcttt aagggttctg tcaactatgc 2460 ttggacaacc gttccaacat atcccacgta ttctttttct ctctctcttc ctgtcttttt 2520 cgatgcaatg taaatttata aaattggaag tccgttttac ttttctatag acgtagatcc 2580 taaattgtc aagaaatgga gaattgactt acaagaaaaa tcaacttctt ttcatttact 2640 attctttttg gtgacaaact ttacttatta tttcgttcta aaatgaaaat ttatttttat 2700 attttaaaat aatttagctt taaactttta attttacttg ttatattttt aataaaaaag 2760 atttattgtc aaataaatgt tgtggccata caataagttt caaattatgt cacaaaaatt 2820 gaaacagagt gagcaaattt ttatttcaag taaactttca aggaattgtg tttaagtttt 2880 ctcaactgat actgatcaat gatgtcttaa atattttgat ttcagcggtg tgatcggtta 2940 tatgctctgc tctactgaag ggccagaagt tgacttcaag aatccagtaa atccaattga 3000 caaagagaca actcaagtca agtccaaatt aggacctctc aagttctaca actctgatgt 3060 aacttcatat ctcacaattt ctttttccgt tttactgtat gttcttcatc aaattttata 3120 actaactctt ttcatattgt cttttttttt cagattcaca aagcagcatt cattttacca 3180 tctttcgcca gaagtatgat cgagtcttaa tcaagtgaat aatgaacact ggtcgtacaa 3240 tcattggacc aagatcaagt cttaatcaag tgaataaata agtgaaatgc aacgtattgt 3300 atgagaattc tgcagtaatt atcataattt ccaattcact aattgttgta aaattctttc 3360 tctgtggtgt ttggtacttt atataaattt ttcctgctga agttttaaaa tggacgttcc 3420 aattcgatcc tcgcaatca gttcattc cttctttcag tgtactaag ccacaatc 3480 tcatagttac cgtggtgttg ttttactat gggatttgcg atcttattaa ggttttgttc 3540 tatgttttgg aattgtaata ttcatatttt taagttactc tgtatctaaa tcggcctagt 3600 ctctctttt tataaatgg cgtcatcgtc gttgcagttt gtttgctcat tttcagaag 3660 tatgatggtt tgaaatgtct ttgttctat ggcctcttta ctgcagctta ccgatttgct 3720 tatgttggat attgcttctg attcatatgg gatatgaagt tggtatttg tgaatgat 3780 aaggaaaaaa tttacaa taactatgcc tcaatcccaa cctattatg atcagcgctt 3840 aaaaaaaaaaaaagtag actaaattt tttaagctta gatgcaatc catacactg 3900 taaaaaaaag taggattc tgttccttc tttagtt aagaagaac tctctgcctt 3960 gtgaatctta ttgcatctct ctaattcagc aaaatattg aacaatgact aggataaat 4020 atttcagcta agataccct aattttctgg atggaatcac cttctccacc aagttgttat 4080 tttcttgcat tgtgagcaga ctagttcaat tacaccatta gagttttttc tcagcactat 4140 cctggcaatg atgcgctaag ctctgaaaac cattagcct tatagagtt ggagctcccc 4200 aaactctt 4210 <210> 3 <211> 3686 <212> DNA <213> Nicotiana tabacum <400> 3 ttgtgagaga ttgtgtcc tctacaatga ttgttgaagt ccctatttat agctatacac 60 aggaaacaaa atcctaggat caagccccctc ttaatgac atatggggt taatgatgaa 120 tatgtagcgg catgacatga atgccaaat tctccgcaac gacttat ttaatattga 180 ggaatatttt ttattaata ctatctggtg acagcattc gttgctcc gttgattacg 240 ttgattttgg gatctactct ataccaccg aagccgttgt ccttgatct cgctttcatt 300 taattcatct tccgtctgcc tccgatttca caagtcatgc acccattca tattattaatg 360 gaaaccaatt ttaccctata siaatggtac atcattcgtc aaatacttta cttggatata 420 aaaaatttg cccgaggagt aacagatgc gagaagaa agcagacgat windowgaatt 480 tttaaaaaag gagagagaaa tgaacacaca catgtacta taaaattagg gtactacttt 540 actaataatt ggacagagac taaattcata ttttagttcc aaaatgtctc gggcagtcca 600 accatgcacg ttgtaatgat ttttactc tattatatcg agttgcgccc tccactccc 660 gtgtccaaa ttgtatataa atgcatatgt gtctattggg agtgtacatc aagctttcat 720 aaagtacaaa tcgtaatact tgttgaaca taatactttc tcttctccaa ttgtttagt 780 ttaattttga aaatggaagt catatctacc aacacaatg gctctaccat cttcaaggt 840 ggtgccattc ccatgaatgg ccaccataat ggcacttcca aacaccaaaa cggccacaag 900 aatgggactt ccgaacaaca gaacgggaca atcagccttg ataatggcaa cgagctactg 960 ggaaactcca attgttaa gcctggttgg tttcagt ggcgcatt atggccaggt 1020 tagtactgag aagaaactc aaatgcatat ttaagttaa attgttagg ctatatag 1080 gagttgatat tcttttagtg atttattaaaaaaaaag tatcaataa attcaaaaaa 1140 tggatagtaa cttcgcatat tactctacac attaatttga aaaatcga attttgcagg 1200 tgaagcattc tcacttaagg ttgagaagtt actgttccag gggaagtctg actaccaaga 1260 1320 tttaatttac ttttacttta ttgcatgtgt acgtacagtc agcaacttat gggaaggttc 1380 tgactttgga tggagcaatt caacacacag agaatggtgg atttccatac actgaaatga 1440 ttgttcatct tccacttggt tccatcccaa acccaaaaaa ggttttgatc atcggcggag 1500 gaattggttt tacattattc gaaatgcttc gttatcctac aatcgaaaaa attgacattg 1560 ttgagatcga tgacgtggta gttgatgtaa gtcaaacttc ttttactcac ataaaaaaat 1620 ggtttagatt gcttcttgtt attttctaa aagaatacta tttttttaaa acaaaatttt 1680 cttttttaca ggtatctaga aaatttttcc cttatctcgc tgctaatttt aacgatcctc 1740 gtgtaaccct agtccttgga gatggtgcgt atttgataat ctcgcttttg tttatcttt 1800 tatttttatt gcatttaatt tttacctttt ggtgtgtggt taattcacct gccattggtt 1860 ctctttcatt tcaggggctg catttgtaaa ggctgcacaa gcagaatatt atgatgctat 1920 tatagtggac tcttctgatc ccattggtac tctattactt cttaatacca agactaatct 1980 tattgaataa gctactaata aacggtaatt gatttctaaa acaatataat ttcaggtcca 2040 gcaaaagatt tgtttgagag gccattcttt gaggcagtag ctaaagccct aaggccagga 2100 ggagttgtat gcacacaggc tgaaagcatt tggcttcata tgcatattat taagcaaatc 2160 attgctaact gtcgtcaagt cttaagggc tctgtcaact atgcttggac tactgttcca 2220 acatatccaa cgtatttttc tctctctctc tcttcctata aaattggaag ttttgattct 2280 ataattgtca agaaatggag aatcagttcc aagaaaaacc aacttcttttt cttttactct 2340 tcaaggtatt gtgtttaatt ttttttcaac tgatatgatc aattattttg atttcagcgg 2400 tgtgattggt tatatgctct gctctactga aggaccagaa attgacttca agaatccagt 2460 aaatccaatt gacaaagaga cagctcaagt caagtccaaa ttagcacctc tcaagttcta 2520 caactctgat gtaacttcat atctcacaat ttctttttc ctattgtact ttatgttctt 2580 cgtcaaattt tataattaac tcttttcaaa ttgtcttttt ttttttcaga ttcacaaagc 2640 agcattcatt ttgccatctt tcgccagaag tatgatcgag tcttaatcaa ctgattaatg 2700 aatactggtg gtacaatcat tggaccaaga tcaataagtg aaagacgtat tgtatgagaa 2760 ttctgcagta attaattatc ataatttcca attaccaat tattgtaaaa ttctttctct 2820 gtggtgtttg gtactttaat ataaatttc ctgcttaagt tttgaatcga cgtatcaact 2880 caatcctcgc aaatcacttc attaccctcc tttcagtgta ctaaagtaaa ctgttgcgga 2940 atatcgtggg ttaactagaa cacaatcaca cacaaatcaa atagagaaga atttattaac 3000 ggggttcggc tagcctaat cctcaggaca aaagcagaga gagttttcca ctatgaatga 3060 gaagaaaaac acaatacaat atataaaatc ctcaactaca accctatat atgatcccaa 3120 aaggtcccaa acatatatga gaaaagtttc ccaatttgac aaagattata ggttttcctt 3180 tcccaaatct attagggcaa tgggttttct taaacctatg gggactatgg ctttcctaaa 3240 aatacaagga aataattcaa accacaaata ataaatcttc cccttggctt gaattctctt 3300 catcaacagg aacaatagct ccttaccttg ccctcaaccc tcgcaagggc tctattgatt 3360 gtcgcacaca tcaaccaagt ctaggcaacg cctaaacttg ttatgagact taatctcttc 3420 agccatagca ctaatccgtc gatttggttc tgtacccgaa atagcttttg gatagctata 3480 acactgaaac acatcagtgg tctctgcaac taccaaagca aatcccacag tattcgtata 3540 tccaagaaga tttccatcaa ctacgtttgc aaacctttgc ggtcggttga tcactctctt 3600 ctctctgtat gttgcaatgc tatatggttg ttgttgcgca ggtgcatcaa cattatcgtc 3660 ttgatcaata ttttgtacct cctcta 3686 <210> 4 <211> 4035 <212> DNA <213> Nicotiana tabacum <400> 4 ggaagagtgt ggtatgggag atgcctccca gggagtacct aaagctgaat actgatggaa 60 gttttaacaa acaaattggg aaagcaggga ttggagggat tctcagagat gaagagggag 120 gctttgtcat ggctttttcg atgcctataa tctataataa catcagtgaa gcagaattga 180 aagccatcaa gtatgggtgt gaatggtgca aatacaaagg aatatcaaac ttcattgtgg 240 aaactgactc gaggatgatc tatgacatac tacagaccaa aaatctaagc aacaacaagt 300 tgaaacaaga gaccgagaaa ttaatggaga ttctggacac ctgcaggaca cctgttaccc 360 attgccttcg cgaagcaaat caagtggcag actggtttgc taaagaggcc accagagcta 420 acgaaggtat cactcataca gattttagac aggtatcaaa agcggccaag ggccctttct 480 tcatggatat gtggcaggtc ccttatttta gaattagata tgaaaaatct aatttttttt 540 tgtaagttaa ttctgtgtat agtgagagga aatcgtctaa tatgtatttt tgcccataga 600 ctcttcctct ccttaggtaa aaaggtagct ccgaggtaag gtttatgttc ccctcagtgt 660 aacctttttt tgtttatata atagacatgg tatgggtcca gctaaacccc caacaccaca 720 ggggatagat acctgggtga ttggtttatt ttttaaaaaa aaaaacttta ctaataattg 780 cacggagaca aaacttatat tttagttcca aaatgacagt ccaaccatgc acgttgtaat 840 gattttttaa ctctattata tcgagttccg ccctccactc ctcggtgtcc aaattgtatt 900 taaatgcata gatatgttta ttgggagtgt acatcaagct ttcagaaaat acaaaccata 960 atactttctc ttctccaatt tgcttagttt aatttggaaa atggaagtca tatctaccaa 1020 cacaaatggc tctactatct tcaagaatgg tgccattccc atgaacggtt accagaatgg 1080 cacttccaaa caccaaaacg gccaccagaa tggcacttcc gaacatcgga acggccacca 1140 gaatgggatt tccgaacacc aaaacggcca ccagaatggc acttccgagc atcagaacgg 1200 ccatcagaat gggacaatca gccatgacaa cggcaacgag ctacagctac tgggaagctc 1260 caactctatt aagcctggtt ggttttcaga gtttagcgca ttatggccag gttagtacta 1320 agaaagaaac tcaaatgcat cgtactcttg tattctgctt tgcgtataat ttagatgatg 1380 gtgtttgact aagcactgag tttaaaaata aaaagtttaa agttaaattg ttactataga 1440 gagctatatc tttaggaact aactaaaaag gaaaaattat cacataaaat tgggatgaag 1500 taagcagtta acttcgcata ttattcgaca cattaatttg aaataaatcg aattttgcag 1560 gtgaagcatt ctcacttaag gttgagaagt tactattcca ggggaagtct gattaccaag 1620 atgtcatgct ctttgaggta attaattaat actaatagtc aagctcatgt atgattat 1680 ttaaagtggt atttttcgtt tatttttaat ttattgcacg tgtacgtaca gtcagcaaca 1740 tatgggaagg ttctgacttt ggatggagca attcacaca cagagaatgg tggattttcca 1800 tacactgaaa tgattgttca tctccactt ggttccatcc caaccctaa aaggttttg atcatcggcg gaggaattgg ttttacatta ttcgaatgc ttcgttatcc tacaatcgaa 1920 aaaattgaca ttgttgagat cgatgacgtg gtagttgatg taagtcaac ttctttact 1980 cacataaaaa atgatttag attcttattt ttctaaaaga attaaaaaaatttccgt 2040 tttacaggta tcagaaaat tttccctta tcttgctgct aatttagcg atcctcgtgt 2100 aaccctagtc cttggagatg gtgcgtattt gataatctcg ttttattt atctttact 2160 tttatttat ttaattttta cctttttgtg tgtggttaat tcacctgcca ttggttctt 2220 ttatttcagg ggctgcattt gtaaaggccg cacaagcagg atattagat gctattatag 2280 tggactctc tgatcccatt tggactctat tactacttaa taccagact attcttatta 2340 aaagctac taataacgt aactctgata gttttctaaa atataat ttcaggtcca 2400 gcaaagact tgtttgagag gccattcttt gaggcagtag ccaagccct aaggccagga 2460 ggagttgtat gcacacaggc tgaaagcatt tggcttcata tgcatattat taagcaaatc 2520 attgctaact gtcgtcaagt ctttaagggc tctgtcaact atgcttggac tactgttcca 2580 acatatccaa cgtatttttc tctctctt cctataaaat tggaagtttt gattctataa 2640 ttgtcaagaa atggagaatc agttccaaga aaaaccaaat tcttttcttt tactcttcaa 2700 ggtgtgttta agttttttaa actgatactg atcaattatt ttgatttcag cggtgtgatt 2760 ggttatatgc tctgttctac tgaaggacca gaagttgact tcaagaatcc agtaaatcca 2820 attgacaaag agacaactca agtcaagtcc aaattagcac ctctcaagtt ctacaactct 2880 gatgtaactt catatctcaa tttcttttt cttatgtac tttatgttct tagtcaaatt 2940 ttataattaa ctcttttcaa attgtctttt tttttcagat tcacaaagca gcattcattt 3000 tgccatcttt cgccagaagt atgatcgagt cttaatcaag tgactaatga atactggcgg 3060 tacaatcatt ggaccaagat cgagtcttaa tcaagtgaat aaataagtga aatgcgacgt 3120 attgtataag aattctgcag tagttaatta tcataatttc caattcacca attactgtaa 3180 aattctttct ctgtggtgtt tggtacttta ctataaattt tcccgcttaa gttttgaatc gacgtttcaa ctcagtcctc gcaaatcact tcattaccct tctttcggta tactaaagta aactgttgca gaatcgtg ggttaactag aacacaatca cacacatagc aatagagaa gaaaaatcaa cacaagatt tattaacgag gttcggctaa gcctaatcct ccgggcaaaa 3480. gcagagaga tttttcacta tgaatgaga gtaaaacaca atacaatcta tagaatttcc 3540. aactacaacc cctatatata gatttcaaaa ggtcccaaac atatatgaga aaggtttccc aatttgacaa ggattatagg ttttcttttc caaaatctat tagggtaatg ggttttccta aacctatgag gactatgggt ttcctaaaaa tacaagga tatttaac caaaataac aaatcttccc cttggtttga attctcttca ttacagga caacagctct ctacctttcc ctcagccctc gcaatggctc tattgattgc cgcactcatc aaccaagtct acgcaacgcc 3780. taaacttgtt aagagactta atctcttcag ccatagcact aatttgtcga tttggttctg tacccgaat agcttttgga actcaaatgc atcagtggtc tctgcaacta ccaaagcaaa tcccacaga ttcgtatatc CAagagatt tccatcaact acatttgcaa 3960 acctttgcgg tcggttgatc actctctc tctctgcatg tgcaatgct atatggttgt 4020 tgttgtgcat gtgca 4035 <210> 5 <211> 4431 <212> DNA <213> Nicotiana tabacum <400> 5 acggtacaat tgaatttgtt gcgtgacttg tagacaagtg aattgatttg tccaaaatga 60 ttaaatc aaatttaaaaaaggctg cgttaaatc aaaggaatg gcaagcctga 120 ctcccggagc aatgctctg aggacagtag taaaacaat atcagacaa aagtaagtt 180 atattatta gcttgaggat aaagtatgtc atcagttttg ttagagattt ggtgtccctct 240 acaatgattg ttgaagtccc tatttatagc tatacatagg aacaagatc ctagaatcaa 300 acccttctta atgacatta atgggagtta ttgatgaata tgtagcggca tgacatgaat 360 gccaaaatttc tccgcaacgg ctattactt atattgagg atatttttc atttaatact 420 atctggtgac aagtattcgt ttgcttccgt tgattgcatt gattttggga tctactatgt 480 accaaccgaa gttgttgtcc ttgatcttcg cttcattta attcatctt cgtcgacctc 540 tgattccaca agtcatgcac ccattcatt atttaatgga aaccaatttt accctgtaca 600 aatggtacaa atactttcct tggataaaa caatttgcc taaggagtaa acgatgcga 660 agtaagaaag crawling aaaaaattt taaaaaagg gagaaagg crawling 720 cgtactata aaattaggggt actactttac taatattgg acagagacta aattcatatt 780 ttagttccaa aatgtctcgg gcagtccaac catgcacgtt gtaatgagtt tttactcta 840 ttatctcgag ttgcgccctc cactccctg tgtccaagtt gtatataaat gcatatatgt 900 ctattgggag tgtacagcga gctttcataa agtacaaatc ataatacttg ttgaacata 960 atactttctc ttctccaatt tgtttagttt aattttgaa atggaagtca tatctaccaa 1020 cacaaatggc 1080 cactccaaa cacctcaacg gctaccagaa cggcactcc aaacaccaaa acggccacca 1140 taatggcact tccgaacatc ggaacggcca ccagaatggg atttccgac accaaacgg 1200 ccaccagaat gggactccg aacatcggaa cggccaccag aatgggatt ccgaacacca 1260 aaacggccac cagaatggga cttccgaaca caccaaacggc caccagaatg ggacttccga 1320 acacagaac gggacaatca gccatgacaa tggcaacgag ctactgggaa actccaactc 1380 tattaagctt ggttggttt cagagtttag cgcattatgg ccaggttagt actgagaaag 1440 aaactcaaat tcatatttaa agttaaaattt gttaggctaa tatagaagt tgattttctt 1500 ttagtgatta atttaaaaag gaaagagtat caaaaatt ccaaaaatg accagtact 1560 tcgcatatta ttctacacat taatttgaa taatcgaat ttgcaggtg aagcattctc 1620 ccttaaggtt gagaagttac tattcagggg gaagtctgac taccagatg tcatgctctt 1680 tgaggtaat atattctaa tacacatgct ttatatga taaatacttt taatttactt 1740 ttagtttatt gcacgtgtac gtacagtcag cacatatgg gaaggttttg actttgtg 1800 gagcaatca accacagag aatggtggat ttccatacac tgaaatgatt gttcatcttc 1860 cactggttc catcccaac ccaaaaagg ttttgatcat cggcggagga attggtttta 1920 cattattcga aatgcttcgt tatcctacaa tcgaaaaaat tgacattgtt gaaatcgatg 1980 acgtggtagt tgatgtaagt caaatttctt ttactcacat aaaaaaatga tttagattgc 2040 ttcttttat ttttctaaaa gaataaat attctctctt agttttaaac aaaattctct 2100 ttcttacagg tatctagaaa atctttccct tatctcgcag ctaattttaa tgatcctcgt 2160 gtaaccctcg ttctcggaga tggtgcgtat ttataatctc gtttttgttt tatcttttat 2220 ttttattca tttaatttac cttttgtgt gtggttaatt tacccgtcat tggttctctt 2280 tcatttcagg ggctgcattt gtaaaggctg cacaagcagg atattatgat gctattatag 2340 tggactcttc tgatcccatt ggtactctat tactacttaa taccaagact aatcttattg 2400 aataagctac tataaactg taattgattt ctaaaataat ataatttcag gtccagcaaa 2460 agatttgttt gagaggccat tctttgaggc agtagccaaa gccctaaggc caggaggagt 2520 tgtatgcaca caggccgaaa gcatttggct tcatatgcat attattaagc aaatcattgc 2580 taactgtcgt caagtcttta agggctctgt caactacgct tggactactg ttccaacata 2640 tcccacgtat tttctctctc tctctcttca tctttgaaaa ttgaaaatcc tgactacttt 2700 ccttcctttg attcctcggt taaaggggcg tagatcataa gattttcaag aaatagataa 2760 tgacgtccaa gaaaaactaa cttcttttca tttactattc tttttggtga caaactttat 2820 ttattatttc gttctaaaga gaaaatttat ttttatattt taaataatt ttgttttaaa 2880 cttttatttt tacttattat atctttaata aaaaaattat agtcaaataa atattatggc 2940 cacactaaac atccaagttt ttgaaaccat aagttttaga gccaaatgag ttaatttgtt 3000 tttggtatgc gggtgcggag tcaaattatg tcacaaaaaat tgtaatggag tgagcaaatt 3060 tttatttcga gtaaactttc aaggtattgt gttaaagttt tttcaactga tactaatcaa 3120 ttatgtctca accattttga tttcagtggt gtaattgggt atatgctctg ctctactgaa 3180 gggccagaag ttgacttcaa gaatccaata aatccaattg acaaagagac aactcaagtc 3240 aagtccaaat tagcacctct caagttttac aattctgatg taacttcata tctaacaatt 3300 tcttttctg ttttactgta tcttcattgt caaaatttta taattaactc ttctcaaatt 3360 atcttttttt ttagattcac aaagcagcat tcattttgcc atctttcgcc agaagtatga 3420 tcgagtctta atcaagtgaa taatgaacac tggtggtgca atcattggac caagatcgag 3480 tcttaatcaa gtgaataaat aagtgaaatg ccgacgtatt gtatgagaat tctacagtaa 3540 ttaattatca taatttccaa ttcaccaatt attgtaaaat tctttctctg tggtgtttgg 3600 tacttcaata taaattttcc tgctgaagtt ttgaatcgat gttccaactc aatcctcgca 3660 aatcagttcc ttatttttct ttcagtgtac ttagtaatct tagtgggatc ggcgcttcca 3720 taaaagaatg taaacttaat atttttttta agcctaaatg caaatccatc acactgtaaa 3780 acatattgat cttcagcctc tgaagatgag tagggatttc tgctcctttc atttacagtc 3840 gagaactttc tgtcttgcat ctctctaatt cagccaaatt attgataacc acgactttag 3900 ttaaacattt gtttaactaa aacaactccc ctaaccatgg taaaacacag tagttatcag 3960 tgtttaatta acctttcctt tttggtaaat atagggtgaa gtcttgctgc gaactttgtt 4020 ctcatatgtt gaagatagta atccaactca agaaacaaac atgattgatt aaatgttgtc 4080 gatgacacac tttcaactga tccaaatggt aacgttccgg ccggctattt tatgtatttg 4140 agtcccgttc tcctacttga tattttccct atgcttgttt gacgttttgt gagttgtgga 4200 gatggttggt ttggttcggg aaggttttgg aatgagtaga gacgcttcaa ctcattttga 4260 aagcttaagt tgtacgagtt gatcaaggtt tgactttggt ataaacgata tcaatttgat 4320 ggtttcagta tgttcatatg gtgattttgg aattagacgc atgttcggat atgaatttgg 4380 aggtttctag aatgttttgt ctctgagtgc caaaagctgg gaatttaaag g 4431 <210> 6 <211> 1128 <212> DNA <213> Nicotiana tabacum <400> 6 atggaagtca tatctaccaa cacaaatggc tctaccatct tcaagaatgg taccattccc 60 atgaacggcc accaaaatgg ctcttccgaa cacctcaacg gctaccagaa tggcatttcc 120 aaacaccaaa acgggcacca gaatggcact tccgaacatc ggaacggcca ccagaatggg 180 acatccgaac aacagaacgg gacaatcagc catgacaatg gcaacgagct actgggaagc 240 tccaactcta ttaagcctgg ttggttttca gagtttagcg cattatggcc aggtgaagca 300 ttctcactta aggtcgagaa gttactattc caggggaaat ctgattacca agatgtcatg 360 ctctttgagt cagcaactta tgggaaggtt ctgactttgg atggagcaat tcaacataca 420 gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg ttccatccca 480 aacccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt cgaaatgctt 540 cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt agttgatgta 600 tccagaaaat ttttccctta tctggcagct aattttaacg atcctcgtgt aaccctagtt 660 ctcggagatg gagctgcatt tgtaaaggct gcacaagcgg gatattatga tgctattata 720 gtggactctt ctgatcccat tggtccagca aaagatttgt ttgagaggcc attctttgag 780 gcagtagcca aagcccttag gccaggagga gttgtatgca cacaggctga aagcatttgg 840 cttcatatgc atattattaa gcaaatcatt gctaactgtc gtcaagtctt taagggttct 900 gtcaactatg cttggacaac cgttccaaca tatcccaccg gtgtgattgg ttatatgctc 960 tgctctactg aagggccaga agttaacttc aagaatccag taaatccaat tgacaaagag 1020 acaactcaag tcaagtccaa attaggacct ctcaagttct acaactctga tattcacaaa 1080 gcagcattca ttttgccatc tttcgcccga agtatgatcg agtcttaa 1128 <210> 7 <211> 1128 <212> DNA <213> Nicotiana tabacum <400> 7 atggaagtca tatctaccaa cacaaatggc tctaccatct tcaagaatgg tgccattccc 60 atgaacggcc accaaaatgg cacttctgaa cacctcaacg gctaccagaa tggcacttcc 120 aaacaccaaa acgggcacca gaatggcact ttcgaacatc ggaacggcca ccagaatggg 180 acatccgaac aacagaacgg gacaatcagc catgacaatg gcaacgagct actgggaagc 240 tccgactcta ttaagcctgg ctggttttca gagtttagcg cattatggcc aggtgaagca 300 ttctcactta aggttgagaa gttactattc caggggaagt ctgattacca agatgtcatg 360 ctctttgagt cagcaactta tgggaaggtt ctgactttgg atggagcaat tcaacataca 420 gagaatggtg gatttccata cactgaaatg attgttcatc taccacttgg ttccatccca 480 aacccaaaaa aggttttgat catcggcgga ggaattggtt ttacattatt cgaaatgctt 540 cgttatcctt caatcgaaaa aattgacatt gttgagatcg atgacgtggt agttgatgta 600 tccagaaaat ttttccctta tctggcagct aattttaacg atcctcgtgt aaccctagtt 660 ctcggagatg gagctgcatt tgtaaaggct gcacaagcgg gatattatga tgctattata 720 gtggactctt ctgatcccat tggtccagca aaagatttgt ttgagaggcc attctttgag 780 gcagtagcca aagcccttag gccaggagga gttgtatgca cacaggctga aagcatttgg 840 cttcatatgc atattattaa gcaaatcatt gctaactgtc gtcaagtctt taagggttct 900 gtcaactatg cttggacaac cgttccaaca tatcccaccg gtgtgatcgg ttatatgctc 960 tgctctactg aagggccaga agttgacttc aagaatccag taaatccaat tgacaaagag 1020 acaactcaag tcaagtccaa attaggacct ctcaagttct acaactctga tattcacaaa 1080 gcagcattca ttttaccatc tttcgccaga agtatgatcg agtcttaa 1128 <210> 8 <211> 1062 <212> DNA <213> Nicotiana tabacum <400> 8 atggaagtca tatctaccaa cacaaatggc tctaccatct tcaagagtgg tgccattccc 60 atgaatggcc accataatgg cacttccaaa caccaaaacg gccacaagaa tgggacttcc 120 gaacaacaga acgggacaat cagccttgat aatggcaacg agctactggg aaactccaat 180 tgtattaagc ctggttggtt ttcagagttt agcgcattat ggccaggtga agcattctca 240 cttaaggttg agaagttact gttccagggg aagtctgact accaagatgt catgctcttt 300 gagtcagcaa cttatgggaa ggttctgact ttggatggag caattcaaca cacagagaat 360 ggtggattc catacactga aatgattgtt catcttccac ttggttccat cccaaaccca 420 aaaaaggttt tgatcatcgg cggaggaatt ggttttacat tattcgaaat gcttcgttat 480 cctacaatcg aaaaattga cattgttgag atcgatgacg tggtagttga tgtatctaga 540 aaattttcc cttatctcgc tgctaatttt aacgatcctc gtgtaaccct agtccttgga 600 gatggggctg catttgtaaa ggctgcacaa gcagaatatt atgatgctat tatagtggac 660 tcttctgatc ccattggtcc agcaaaagat ttgtttgaga ggccattctt tgaggcagta 720 gctaaagccc taaggccagg aggagttgta tgcacacagg ctgaaagcat ttggcttcat 780 atgcatatta ttaagcaaat cattgctaac tgtcgtcaag tctttaaggg ctctgtcaac 840 tatgcttgga ctactgttcc aacatatcca accggtgtga ttggttatat gctctgctct 900 actgaaggac cagaaattga cttcaagaat ccagtaaatc caattgacaa agagacagct 960 caagtcaagt ccaaattagc acctctcaag ttctacaact ctgatattca caaagcagca 1020 ttcattttgc catctttcgc cagaagtatg atcgagtctt aa 1062 <210> 9 <211> 1146 <212> DNA <213> Nicotiana tabacum <400> 9 atggaagtca tatctaccaa cacaaatggc tctactatct tcaagaatgg tgccattccc 60 atgaacggtt accagaatgg cacttccaaa caccaaaacg gccaccagaa tggcacttcc 120 gaacatcgga acggccacca gaatgggatt tccgaacacc aaaacggcca ccagaatggc 180 acttccgagc atcagaacgg ccatcagaat gggacaatca gccatgacaa cggcaacgag 240 ctacagctac tgggaagctc caactctatt aagcctggtt ggttttcaga gtttagcgca 300 ttatggccag gtgaagcatt ctcacttaag gttgagaagt tactattcca ggggaagtct 360 gattaccaag atgtcatgct ctttgagtca gcaacatatg ggaaggttct gactttggat 420 ggagcaattc aacacacaga gaatggtgga tttccataca ctgaaatgat tgttcatctt 480 ccacttggtt ccatcccaaa ccctaaaaag gttttgatca tcggcggagg aattggtttt 540 acattattcg aaatgcttcg ttatcctaca atcgaaaaaa ttgacattgt tgagatcgat 600 gacgtggtag ttgatgtatc tagaaaattt ttcccttatc ttgctgctaa ttttagcgat 660 cctcgtgtaa ccctagtcct tggagatggg gctgcatttg taaaggccgc acaagcagga 720 tattatgatg ctattatagt ggactcttct gatcccattg gtccagcaaa agacttgttt 780 gagaggccat tctttgaggc agtagccaaa gccctaaggc caggaggagt tgtatgcaca 840 caggctgaaa gcatttggct tcatatgcat attattaagc aaatcattgc taactgtcgt 900 caagtcttta agggctctgt caactatgct tggactactg ttccaacata tccaaccggt 960 gtgattggtt atatgctctg ttctactgaa ggaccagaag ttgacttcaa gaatccagta 1020 aatccaattg acaaagagac aactcaagtc aagtccaaat tagcacctct caagttctac 1080 aactctgata ttcacaaagc agcattcatt ttgccatctt tcgccagaag tatgatcgag 1140 tcttaa 1146 <210> 10 <211> 1260 <212> DNA <213> Nicotiana tabacum <400> 10 atggaagtca tatctaccaa cacaaatggc tcgaccatct tcaagaatgg tgccattccc 60 atgaatggcc accagagtgg cacttccaaa cacctcaacg gctaccagaa cggcacttcc 120 aaacaccaaa acggccacca taatggcact tccgaacatc ggaacggcca ccagaatggg 180 atttccgaac accaaaacgg ccaccagaat gggacttccg aacatcggaa cggccaccag 240 aatgggattt ccgaacacca aaacggccac cagaatggga cttccgaaca ccaaaacggc 300 caccagaatg ggacttccga acaacagaac gggacaatca gccatgacaa tggcaacgag 360 ctactgggaa actccaactc tattaagctt ggttggtttt cagagtttag cgcattatgg 420 ccaggtgaag cattctccct taaggttgag aagttactat ttcaggggaa gtctgactac 480 caagatgtca tgctctttga gtcagcaaca tatgggaagg ttttgacttt ggatggagca 540 attcaacaca cagagaatgg tggatttcca tacactgaaa tgattgttca tcttccactt 600 ggttccatcc caaacccaaa aaaggttttg atcatcggcg gaggaattgg ttttacatta 660 ttcgaaatgc ttcgttatcc tacaatcgaa aaaattgaca ttgttgaaat cgatgacgtg 720 gtagttgatg tatctagaaa atctttccct tatctcgcag ctaattttaa tgatcctcgt 780 gtaaccctcg ttctcggaga tggggctgca tttgtaaagg ctgcacaagc aggatattat 840 gatgctatta tagtggactc ttctgatccc attggtccag caaaagattt gtttgagagg 900 ccattctttg aggcagtagc caaagcccta aggccaggag gagttgtatg cacacaggcc 960 gaaagcattt ggcttcatat gcatattatt aagcaaatca ttgctaactg tcgtcaagtc 1020 tttaagggct ctgtcaacta cgcttggact actgttccaa catatcccac tggtgtaatt 1080 gggtatatgc tctgctctac tgaagggcca gaagttgact tcaagaatcc aataaatcca 1140 attgacaaag agacaactca agtcaagtcc aaattagcac ctctcaagtt ttacaattct 1200 gatattcaca aagcagcatt cattttgcca tctttcgcca gaagtatgat cgagtcttaa 1260 <210> 11 <211> 375 <212> PRT <213> Nicotiana tabacum <400> 11 Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn 1 5 10 15 Gly Thr Ile Pro Met Asn Gly His Gln Asn Gly Ser Ser Glu His Leu 20 25 30 Asn Gly Tyr Gln Asn Gly Ile Ser Lys His Gln Asn Gly His Gln Asn 35 40 45 Gly Thr Ser Glu His Arg Asn Gly His Gln Asn Gly Thr Ser Glu Gln 50 55 60 Gln Asn Gly Thr Ile Ser His Asp Asn Gly Asn Glu Leu Leu Gly Ser 65 70 75 80 Ser Asn Ser Ile Lys Pro Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp 85 90 95 Pro Gly Glu Ala Phe Ser Leu Lys Val Glu Lys Leu Leu Phe Gln Gly 100 105 110 Lys Ser Asp Tyr Gln Asp Val Met Leu Phe Glu Ser Ala Thr Tyr Gly 115 120 125 Lys Val Leu Thr Leu Asp Gly Ala Ile Gln His Thr Glu Asn Gly Gly 130 135 140 Phe Pro Tyr Thr Glu Met Ile Val His Leu Pro Leu Gly Ser Ile Pro 145 150 155 160 Asn Pro Lys Lys Val Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu 165 170 175 Phe Glu Met Leu Arg Tyr Pro Ser Ile Glu Lys Ile Asp Ile Val Glu 180 185 190 Ile Asp Asp Val Val Val Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu 195 200 205 Ala Ala Asn Phe Asn Asp Pro Arg Val Thr Leu Val Leu Gly Asp Gly 210 215 220 Ala Ala Phe Val Lys Ala Ala Gln Ala Gly Tyr Tyr Asp Ala Ile Ile 225 230 235 240 Val Asp Ser Ser Asp Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg 245 250 255 Pro Phe Phe Glu Ala Val Ala Lys Ala Leu Arg Pro Gly Gly Val Val 260 265 270 Cys Thr Gln Ala Glu Ser Ile Trp Leu His Met His Ile Ile Lys Gln 275 280 285 Ile Ile Ala Asn Cys Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala 290 295 300 Trp Thr Thr Val Pro Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu 305 310 315 320 Cys Ser Thr Glu Gly Pro Glu Val Asn Phe Lys Asn Pro Val Asn Pro 325 330 335 Ile Asp Lys Glu Thr Thr Gln Val Lys Ser Lys Leu Gly Pro Leu Lys 340 345 350 Phe Tyr Asn Ser Asp Ile His Lys Ala Ala Phe Ile Leu Pro Ser Phe 355 360 365 Ala Arg Ser Met Ile Glu Ser 370 375 <210> 12 <211> 375 <212> PRT <213> Nicotiana tabacum <400> 12 Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn 1 5 10 15 Gly Ala Ile Pro Met Asn Gly His Gln Asn Gly Thr Ser Glu His Leu 20 25 30 Asn Gly Tyr Gln Asn Gly Thr Ser Lys His Gln Asn Gly His Gln Asn 35 40 45 Gly Thr Phe Glu His Arg Asn Gly His Gln Asn Gly Thr Ser Glu Gln 50 55 60 Gln Asn Gly Thr Ile Ser His Asp Asn Gly Asn Glu Leu Leu Gly Ser 65 70 75 80 Ser Asp Ser Ile Lys Pro Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp 85 90 95 Pro Gly Glu Ala Phe Ser Leu Lys Val Glu Lys Leu Leu Phe Gln Gly 100 105 110 Lys Ser Asp Tyr Gln Asp Val Met Leu Phe Glu Ser Ala Thr Tyr Gly 115 120 125 Lys Val Leu Thr Leu Asp Gly Ala Ile Gln His Thr Glu Asn Gly Gly 130 135 140 Phe Pro Tyr Thr Glu Met Ile Val His Leu Pro Leu Gly Ser Ile Pro 145 150 155 160 Asn Pro Lys Lys Val Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu 165 170 175 Phe Glu Met Leu Arg Tyr Pro Ser Ile Glu Lys Ile Asp Ile Val Glu 180 185 190 Ile Asp Asp Val Val Val Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu 195 200 205 Ala Ala Asn Phe Asn Asp Pro Arg Val Thr Leu Val Leu Gly Asp Gly 210 215 220 Ala Ala Phe Val Lys Ala Ala Gln Ala Gly Tyr Tyr Asp Ala Ile Ile 225 230 235 240 Val Asp Ser Ser Asp Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg 245 250 255 Pro Phe Phe Glu Ala Val Ala Lys Ala Leu Arg Pro Gly Gly Val Val 260 265 270 Cys Thr Gln Ala Glu Ser Ile Trp Leu His Met His Ile Ile Lys Gln 275 280 285 Ile Ile Ala Asn Cys Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala 290 295 300 Trp Thr Thr Val Pro Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu 305 310 315 320 Cys Ser Thr Glu Gly Pro Glu Val Asp Phe Lys Asn Pro Val Asn Pro 325 330 335 Ile Asp Lys Glu Thr Thr Gln Val Lys Ser Lys Leu Gly Pro Leu Lys 340 345 350 Phe Tyr Asn Ser Asp Ile His Lys Ala Ala Phe Ile Leu Pro Ser Phe 355 360 365 Ala Arg Ser Met Ile Glu Ser 370 375 <210> 13 <211> 353 <212> PRT <213> Nicotiana tabacum <400> 13 Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Ser 1 5 10 15 Gly Ala Ile Pro Met Asn Gly His His Asn Gly Thr Ser Lys His Gln 20 25 30 Asn Gly His Lys Asn Gly Thr Ser Glu Gln Gln Asn Gly Thr Ile Ser 35 40 45 Leu Asp Asn Gly Asn Glu Leu Leu Gly Asn Ser Asn Cys Ile Lys Pro 50 55 60 Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp Pro Gly Glu Ala Phe Ser 65 70 75 80 Leu Lys Val Glu Lys Leu Leu Phe Gln Gly Lys Ser Asp Tyr Gln Asp 85 90 95 Val Met Leu Phe Glu Ser Ala Thr Tyr Gly Lys Val Leu Thr Leu Asp 100 105 110 Gly Ala Ile Gln His Thr Glu Asn Gly Gly Phe Pro Tyr Thr Glu Met 115 120 125 Ile Val His Leu Pro Leu Gly Ser Ile Pro Asn Pro Lys Lys Val Leu 130 135 140 Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu Phe Glu Met Leu Arg Tyr 145 150 155 160 Pro Thr Ile Glu Lys Ile Asp Ile Val Glu Ile Asp Asp Val Val Val 165 170 175 Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu Ala Ala Asn Phe Asn Asp 180 185 190 Pro Arg Val Thr Leu Val Leu Gly Asp Gly Ala Ala Phe Val Lys Ala 195 200 205 Ala Gln Ala Glu Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser Asp Pro 210 215 220 Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu Ala Val 225 230 235 240 Ala Lys Ala Leu Arg Pro Gly Gly Val Val Cys Thr Gln Ala Glu Ser 245 250 255 Ile Trp Leu His Met His Ile Ile Lys Gln Ile Ile Ala Asn Cys Arg 260 265 270 Gln Val Phe Lys Gly Ser Val Asn Tyr Ala Trp Thr Thr Val Pro Thr 275 280 285 Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu Cys Ser Thr Glu Gly Pro 290 295 300 Glu Ile Asp Phe Lys Asn Pro Val Asn Pro Ile Asp Lys Glu Thr Ala 305 310 315 320 Gln Val Lys Ser Lys Leu Ala Pro Leu Lys Phe Tyr Asn Ser Asp Ile 325 330 335 His Lys Ala Ala Phe Ile Leu Pro Ser Phe Ala Arg Ser Met Ile Glu 340 345 350 Ser <210> 14 <211> 381 <212> PRT <213> Nicotiana tabacum <400> 14 Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn 1 5 10 15 Gly Ala Ile Pro Met Asn Gly Tyr Gln Asn Gly Thr Ser Lys His Gln 20 25 30 Asn Gly His Gln Asn Gly Thr Ser Glu His Arg Asn Gly His Gln Asn 35 40 45 Gly Ile Ser Glu His Gln Asn Gly His Gln Asn Gly Thr Ser Glu His 50 55 60 Gln Asn Gly His Gln Asn Gly Thr Ile Ser His Asp Asn Gly Asn Glu 65 70 75 80 Leu Gln Leu Leu Gly Ser Ser Asn Ser Ile Lys Pro Gly Trp Phe Ser 85 90 95 Glu Phe Ser Ala Leu Trp Pro Gly Glu Ala Phe Ser Leu Lys Val Glu 100 105 110 Lys Leu Leu Phe Gln Gly Lys Ser Asp Tyr Gln Asp Val Met Leu Phe 115 120 125 Glu Ser Ala Thr Tyr Gly Lys Val Leu Thr Leu Asp Gly Ala Ile Gln 130 135 140 His Thr Glu Asn Gly Gly Phe Pro Tyr Thr Glu Met Ile Val His Leu 145 150 155 160 Pro Leu Gly Ser Ile Pro Asn Pro Lys Lys Val Leu Ile Ile Gly Gly 165 170 175 Gly Ile Gly Phe Thr Leu Phe Glu Met Leu Arg Tyr Pro Thr Ile Glu 180 185 190 Lys Ile Asp Ile Val Glu Ile Asp Asp Val Val Val Asp Val Ser Arg 195 200 205 Lys Phe Phe Pro Tyr Leu Ala Ala Asn Phe Ser Asp Pro Arg Val Thr 210 215 220 Leu Val Leu Gly Asp Gly Ala Ala Phe Val Lys Ala Ala Gln Ala Gly 225 230 235 240 Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser Asp Pro Ile Gly Pro Ala 245 250 255 Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu Ala Val Ala Lys Ala Leu 260 265 270 Arg Pro Gly Gly Val Val Cys Thr Gln Ala Glu Ser Ile Trp Leu His 275 280 285 Met His Ile Ile Lys Gln Ile Ile Ala Asn Cys Arg Gln Val Phe Lys 290 295 300 Gly Ser Val Asn Tyr Ala Trp Thr Thr Val Pro Thr Tyr Pro Thr Gly 305 310 315 320 Val Ile Gly Tyr Met Leu Cys Ser Thr Glu Gly Pro Glu Val Asp Phe 325 330 335 Lys Asn Pro Val Asn Pro Ile Asp Lys Glu Thr Thr Gln Val Lys Ser 340 345 350 Lys Leu Ala Pro Leu Lys Phe Tyr Asn Ser Asp Ile His Lys Ala Ala 355 360 365 Phe Ile Leu Pro Ser Phe Ala Arg Ser Met Ile Glu Ser 370 375 380 <210> 15 <211> 419 <212> PRT <213> Nicotiana tabacum <400> 15 Met Glu Val Ile Ser Thr Asn Thr Asn Gly Ser Thr Ile Phe Lys Asn 1 5 10 15 Gly Ala Ile Pro Met Asn Gly His Gln Ser Gly Thr Ser Lys His Leu 20 25 30 Asn Gly Tyr Gln Asn Gly Thr Ser Lys His Gln Asn Gly His His Asn 35 40 45 Gly Thr Ser Glu His Arg Asn Gly His Gln Asn Gly Ile Ser Glu His 50 55 60 Gln Asn Gly His Gln Asn Gly Thr Ser Glu His Arg Asn Gly His Gln 65 70 75 80 Asn Gly Ile Ser Glu His Gln Asn Gly His Gln Asn Gly Thr Ser Glu 85 90 95 His Gln Asn Gly His Gln Asn Gly Thr Ser Glu Gln Gln Asn Gly Thr 100 105 110 Ile Ser His Asp Asn Gly Asn Glu Leu Leu Gly Asn Ser Asn Ser Ile 115 120 125 Lys Leu Gly Trp Phe Ser Glu Phe Ser Ala Leu Trp Pro Gly Glu Ala 130 135 140 Phe Ser Leu Lys Val Glu Lys Leu Leu Phe Gln Gly Lys Ser Asp Tyr 145 150 155 160 Gln Asp Val Met Leu Phe Glu Ser Ala Thr Tyr Gly Lys Val Leu Thr 165 170 175 Leu Asp Gly Ala Ile Gln His Thr Glu Asn Gly Gly Phe Pro Tyr Thr 180 185 190 Glu Met Ile Val His Leu Pro Leu Gly Ser Ile Pro Asn Pro Lys Lys 195 200 205 Val Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu Phe Glu Met Leu 210 215 220 Arg Tyr Pro Thr Ile Glu Lys Ile Asp Ile Val Glu Ile Asp Asp Val 225 230 235 240 Val Val Asp Val Ser Arg Lys Ser Phe Pro Tyr Leu Ala Ala Asn Phe 245 250 255 Asn Asp Pro Arg Val Thr Leu Val Leu Gly Asp Gly Ala Ala Phe Val 260 265 270 Lys Ala Ala Gln Ala Gly Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser 275 280 285 Asp Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu 290 295 300 Ala Val Ala Lys Ala Leu Arg Pro Gly Gly Val Val Cys Thr Gln Ala 305 310 315 320 Glu Ser Ile Trp Leu His Met His Ile Ile Lys Gln Ile Ile Ala Asn 325 330 335 Cys Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala Trp Thr Thr Val 340 345 350 Pro Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu Cys Ser Thr Glu 355 360 365 Gly Pro Glu Val Asp Phe Lys Asn Pro Ile Asn Pro Ile Asp Lys Glu 370 375 380 Thr Thr Gln Val Lys Ser Lys Leu Ala Pro Leu Lys Phe Tyr Asn Ser 385 390 395 400 Asp Ile His Lys Ala Ala Phe Ile Leu Pro Ser Phe Ala Arg Ser Met 405 410 415 Ile Glu Ser <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 cccatgaacg gccaccaaaa 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 17 ggcacttcca aacaccaaaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 gttgttcgga tgtcccattc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 19 ctaaactctg aaaaccaacc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 20 tttcagagtt tagcgcatta 20 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 21 gatggagcaa ttcaacatac aga 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 gatggagcaa ttcaacacac aga 23 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 23 tggcatttcc aaacaccaaa cgggcaccag aatggcactt 40 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 24 ccaactctat taagcctggt ggttttcaga gtttagcgca 40 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 ttctgacttt ggatggagca atacagagaa tggtggattt 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 26 ctgactttgg atggagcaat gagaatggtg gatttccata 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 27 tgactttgga tggagcaatt cagagaatgg tggatttcca 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 28 tgactttgga tggagcaatt agaatggtgg atttccatac 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 29 gactttggat ggagcaattc agagaatggt ggatttccat 40 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 30 gactttggat ggagcaattc gagaatggtg gatttccata 40 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 31 gactttggat ggagcaattc agaatggtgg atttccatac 40 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 32 gactttggat ggagcaattc tggatttcca tacactgaaa 40 <210> 33 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 33 actttggatg gagcaattca tacagagaat ggtggatttc c 41 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 34 actttggatg gagcaattca cagagaatgg tggatttcca 40 <210> 35 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 35 actttggatg gagcaattca agagaatggt ggatttccat 40 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 36 actttggatg gagcaattca gagaatggtg gatttccata 40 <210> 37 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 37 actttggatg gagcaattca atacactgaa atgattgttc 40 <210> 38 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 38 ctttggatgg agcaattcaa tacagagaat ggtggatttc 40 <210> 39 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 39 ctttggatgg agcaattcaa cagagaatgg tggatttcca 40 <210> 40 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 40 ctttggatgg agcaattcaa gagaatggtg gatttccata 40 <210> 41 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 41 ctttggatgg agcaattcaa agaatggtgg atttccatac 40 <210> 42 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 42 tttggatgga gcaattcaac tacagagaat ggtggatttc 40 <210> 43 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 43 tttggatgga gcaattcaac cagagaatgg tggatttcca 40 <210> 44 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 44 tttggatgga gcaattcaac agagaatggt ggatttccat 40 <210> 45 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 45 tttggatgga gcaattcaac gagaatggtg gatttccata 40 <210> 46 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 46 tttggatgga gcaattcaac agaatggtgg atttccatac 40 <210> 47 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 47 tttggatgga gcaattcaac aatggtggat ttccatacac 40 <210> 48 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 48 ttggatggag caattcaaca cagagaatgg tggatttcca 40 <210> 49 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 49 ttggatggag caattcaaca gagaatggtg gatttccata 40 <210> 50 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 50 tggatggagc aattcaacat agagaatggt ggatttccat 40 <210> 51 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 51 tggatggagc aattcaacat gagaatggtg gatttccata 40 <210> 52 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 52 tggatggagc aattcaacat agaatggtgg atttccatac 40 <210> 53 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 53 tggatggagc aattcaacat aatggtggat ttccatacac 40 <210> 54 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 54 ggatggagca attcaacata gagaatggtg gatttccata 40 <210> 55 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 55 caattcaaca tacagagaat gtggatttcc atacactgaa 40 <210> 56 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 56 gcacttccaa acaccaaaac agggcaccag aatggcactt t 41 <210> 57 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 57 ttctgacttt ggatggagca atacagagaa tggtggattt 40 <210> 58 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 58 ttctgacttt ggatggagca gagaatggtg gatttccata 40 <210> 59 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 59 ctgactttgg atggagcaat acagagaatg gtggatttcc 40 <210> 60 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 60 ctgactttgg atggagcaat gagaatggtg gatttccata 40 <210> 61 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 61 tgactttgga tggagcaatt cagagaatgg tggatttcca 40 <210> 62 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 62 tgactttgga tggagcaatt agaatggtgg atttccatac 40 <210> 63 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 63 gactttggat ggagcaattc agagaatggt ggatttccat 40 <210> 64 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 64 gactttggat ggagcaattc gagaatggtg gatttccata 40 <210> 65 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 65 gactttggat ggagcaattc agaatggtgg atttccatac 40 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 66 gactttggat ggagcaattc gaatggtgga tttccataca 40 <210> 67 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 67 gactttggat ggagcaattc tggatttcca tacactgaaa 40 <210> 68 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 68 actttggatg gagcaattca atacagagaa tggtggattt 40 <210> 69 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 69 actttggatg gagcaattca acagagaatg gtggatttcc 40 <210> 70 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 70 actttggatg gagcaattca agagaatggt ggatttccat 40 <210> 71 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 71 actttggatg gagcaattca gagaatggtg gatttccata 40 <210> 72 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 72 actttggatg gagcaattca agaatggtgg atttccatac 40 <210> 73 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 73 actttggatg gagcaattca aatggtggat ttccatacac 40 <210> 74 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 74 ctttggatgg agcaattcaa atacagagaa tggtggattt 40 <210> 75 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 75 ctttggatgg agcaattcaa tacagagaat ggtggatttc 40 <210> 76 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 76 ctttggatgg agcaattcaa cagagaatgg tggatttcca 40 <210> 77 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 77 ctttggatgg agcaattcaa agagaatggt ggatttccat 40 <210> 78 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 78 ctttggatgg agcaattcaa agaatggtgg atttccatac 40 <210> 79 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 79 tttggatgga gcaattcaac tacagagaat ggtggatttc c 41 <210> 80 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 80 tttggatgga gcaattcaac agagaatggt ggatttccat 40 <210> 81 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 81 tttggatgga gcaattcaac gagaatggtg gatttccata 40 <210> 82 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 82 tttggatgga gcaattcaac agaatggtgg atttccatac 40 <210> 83 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 83 tttggatgga gcaattcaac gaatggtgga tttccataca 40 <210> 84 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 84 tttggatgga gcaattcaac ggatttccat acactgaaat 40 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 85 ttggatggag caattcaaca acagagaatg gtggatttcc 40 <210> 86 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 86 ttggatggag caattcaaca cagagaatgg tggatttcca 40 <210> 87 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 87 ttggatggag caattcaaca agagaatggt ggatttccat 40 <210> 88 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 88 ttggatggag caattcaaca gagaatggtg gatttccata 40 <210> 89 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 89 ttggatggag caattcaaca agaatggtgg atttccatac 40 <210> 90 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 90 ttggatggag caattcaaca gaatggtgga tttccataca 40 <210> 91 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 91 ttggatggag caattcaaca aatggtggat ttccatacac 40 <210> 92 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 92 tggatggagc aattcaacat agagaatggt ggatttccat 40 <210> 93 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 93 tggatggagc aattcaacat agaatggtgg atttccatac 40 <210> 94 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 94 tggatggagc aattcaacat atggtggatt tccatacact 40 <210> 95 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 95 ggatggagca attcaacata gagaatggtg gatttccata 40 <210> 96 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 96 ggatggagca attcaacata agaatggtgg atttccatac 40 <210> 97 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 97 gagcaattca acatacagag tggtggattt ccatacactg 40 <210> 98 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 98 tggcacttcc aaacaccaaa cggccacaag aatgggactt 40 <210> 99 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 99 ccaattgtat taagcctggt ggttttcaga gtttagcgca 40 <210> 100 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 100 tgactttgga tggagcaatt cacagagaat ggtggatttc 40 <210> 101 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 101 tgactttgga tggagcaatt cagagaatgg tggatttcca 40 <210> 102 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 102 tgactttgga tggagcaatt agaatggtgg atttccatac 40 <210> 103 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 103 gactttggat ggagcaattc gagaatggtg gatttccata 40 <210> 104 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 104 gactttggat ggagcaattc aatggtggat ttccatacac 40 <210> 105 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 105 actttggatg gagcaattca acacagagaa tggtggattt 40 <210> 106 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 106 actttggatg gagcaattca acagagaatg gtggatttcc 40 <210> 107 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 107 actttggatg gagcaattca cagagaatgg tggatttcca 40 <210> 108 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 108 actttggatg gagcaattca agagaatggt ggatttccat 40 <210> 109 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 109 actttggatg gagcaattca gagaatggtg gatttccata 40 <210> 110 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 110 actttggatg gagcaattca agaatggtgg atttccatac 40 <210> 111 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 111 actttggatg gagcaattca aatggtggat ttccatacac 40 <210> 112 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 112 ctttggatgg agcaattcaa acacagagaa tggtggattt 40 <210> 113 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 113 ctttggatgg agcaattcaa cacagagaat ggtggatttc 40 <210> 114 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 114 ctttggatgg agcaattcaa cagagaatgg tggatttcca 40 <210> 115 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 115 ctttggatgg agcaattcaa agaatggtgg atttccatac 40 <210> 116 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 116 tttggatgga gcaattcaac acagagaatg gtggatttcc 40 <210> 117 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 117 tttggatgga gcaattcaac agagaatggt ggatttccat 40 <210> 118 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 118 tttggatgga gcaattcaac gagaatggtg gatttccata 40 <210> 119 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 119 tttggatgga gcaattcaac agaatggtgg atttccatac 40 <210> 120 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 120 tttggatgga gcaattcaac aatggtggat ttccatacac 40 <210> 121 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 121 tttggatgga gcaattcaac ggatttccat acactgaaat 40 <210> 122 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 122 ttggatggag caattcaaca cagagaatgg tggatttcca 40 <210> 123 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 123 ttggatggag caattcaaca gagaatggtg gatttccata 40 <210> 124 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 124 ttggatggag caattcaaca gaatggtgga tttccataca 40 <210> 125 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 125 ttggatggag caattcaaca aatggtggat ttccatacac 40 <210> 126 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 126 ttggatggag caattcaaca tggatttcca tacactgaaa 40 <210> 127 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 127 tggatggagc aattcaacac gagaatggtg gatttccata 40 <210> 128 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 128 tggatggagc aattcaacac agaatggtgg atttccatac 40 <210> 129 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 129 ggatggagca attcaacaca gagaatggtg gatttccata 40 <210> 130 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 130 ggatggagca attcaacaca gaatggtgga tttccataca 40 <210> 131 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 131 ggatggagca attcaacaca gtggatttcc atacactgaa 40 <210> 132 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 132 gatggagcaa ttcaacacac tggtggattt ccatacactg 40 <210> 133 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 133 tggcacttcc aaacaccaaa cggccaccag aatggcactt 40 <210> 134 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 134 ccaactctat taagcctggt ggttttcaga gtttagcgca 40 <210> 135 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 135 aacatatggg aaggttctga ttggatggag caattcaaca 40 <210> 136 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 136 atgggaaggt tctgactttg tggagcaatt caacacacag 40 <210> 137 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 137 gttctgactt tggatggagc ttcaacacac agagaatggt 40 <210> 138 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 138 ctgactttgg atggagcaat acagagaatg gtggatttcc 40 <210> 139 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 139 ctgactttgg atggagcaat gagaatggtg gatttccata 40 <210> 140 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 140 ctgactttgg atggagcaat agaatggtgg atttccatac 40 <210> 141 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 141 tgactttgga tggagcaatt cagagaatgg tggatttcca 40 <210> 142 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 142 gactttggat ggagcaattc acacacagag aatggtggat 40 <210> 143 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 143 gactttggat ggagcaattc cacacagaga atggtggatt 40 <210> 144 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 144 gactttggat ggagcaattc gagaatggtg gatttccata 40 <210> 145 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 145 gactttggat ggagcaattc aatggtggat ttccatacac 40 <210> 146 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 146 actttggatg gagcaattca acacagagaa tggtggattt 40 <210> 147 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 147 actttggatg gagcaattca acagagaatg gtggatttcc 40 <210> 148 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 148 actttggatg gagcaattca agagaatggt ggatttccat 40 <210> 149 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 149 actttggatg gagcaattca agaatggtgg atttccatac 40 <210> 150 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 150 actttggatg gagcaattca aatggtggat ttccatacac 40 <210> 151 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 151 actttggatg gagcaattca tggatttcca tacactgaaa 40 <210> 152 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 152 actttggatg gagcaattca gatttccata cactgaaatg 40 <210> 153 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 153 ctttggatgg agcaattcaa acacagagaa tggtggattt 40 <210> 154 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 154 ctttggatgg agcaattcaa cagagaatgg tggatttcca 40 <210> 155 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 155 ctttggatgg agcaattcaa agagaatggt ggatttccat 40 <210> 156 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 156 ctttggatgg agcaattcaa agaatggtgg atttccatac 40 <210> 157 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 157 tttggatgga gcaattcaac acagagaatg gtggatttcc 40 <210> 158 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 158 tttggatgga gcaattcaac agagaatggt ggatttccat 40 <210> 159 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 159 tttggatgga gcaattcaac gagaatggtg gatttccata 40 <210> 160 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 160 ttggatggag caattcaaca cagagaatgg tggatttcca 40 <210> 161 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 161 ttggatggag caattcaaca gagaatggtg gatttccata 40 <210> 162 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 162 ttggatggag caattcaaca agaatggtgg atttccatac 40 <210> 163 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 163 ttggatggag caattcaaca ggatttccat acactgaaat 40 <210> 164 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 164 tggatggagc aattcaacac agagaatggt ggatttccat 40 <210> 165 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 165 tggatggagc aattcaacac agaatggtgg atttccatac 40 <210> 166 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 166 tggatggagc aattcaacac tggtggattt ccatacactg 40 <210> 167 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 167 ggatggagca attcaacaca gagaatggtg gatttccata 40 <210> 168 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 168 tggagcaatt caacacacag atggtggatt tccatacact 40 <210> 169 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 169 tggagcaatt caacacacag tggtggattt ccatacactg 40 <210> 170 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 170 ggagcaattc aacacacaga aatggtggat ttccatacac 40 <210> 171 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 171 caattcaaca cacagagaat atttccatac actgaaatga 40 <210> 172 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 172 aacacacaga gaatggtgga aaatgattgt tcatcttcca 40 <210> 173 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 173 cggcacttcc aaacaccaaa cggccaccat aatggcactt 40 <210> 174 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 174 ttttgacttt ggatggagca agagaatggt ggatttccat 40 <210> 175 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 175 ttgactttgg atggagcaat gaatggtgga tttccataca 40 <210> 176 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 176 tgactttgga tggagcaatt cagagaatgg tggatttcca 40 <210> 177 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 177 tgactttgga tggagcaatt agaatggtgg atttccatac 40 <210> 178 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 178 gactttggat ggagcaattc aatggtggat ttccatacac 40 <210> 179 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 179 gactttggat ggagcaattc tggtggattt ccatacactg 40 <210> 180 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 180 actttggatg gagcaattca acacagagaa tggtggattt 40 <210> 181 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 181 actttggatg gagcaattca acagagaatg gtggatttcc 40 <210> 182 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 182 actttggatg gagcaattca agagaatggt ggatttccat 40 <210> 183 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 183 actttggatg gagcaattca gagaatggtg gatttccata 40 <210> 184 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 184 actttggatg gagcaattca agaatggtgg atttccatac 40 <210> 185 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 185 ctttggatgg agcaattcaa cagagaatgg tggatttcca 40 <210> 186 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 186 ctttggatgg agcaattcaa agagaatggt ggatttccat 40 <210> 187 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 187 tttggatgga gcaattcaac acagagaatg gtggatttcc 40 <210> 188 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 188 tttggatgga gcaattcaac agagaatggt ggatttccat 40 <210> 189 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 189 tttggatgga gcaattcaac gagaatggtg gatttccata 40 <210> 190 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 190 ttggatggag caattcaaca gagaatggtg gatttccata 40 <210> 191 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 191 ttggatggag caattcaaca agaatggtgg atttccatac 40 <210> 192 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 192 tggatggagc aattcaacac agagaatggt ggatttccat 40 <210> 193 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 193 tggatggagc aattcaacac gagaatggtg gatttccata 40 <210> 194 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 194 tggatggagc aattcaacac agaatggtgg atttccatac 40 <210> 195 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 195 tggatggagc aattcaacac atggtggatt tccatacact 40 <210> 196 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 196 ggatggagca attcaacaca gagaatggtg gatttccata 40 <210> 197 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 197 tggagcaatt caacacacag gaatggtgga tttccataca 40 <210> 198 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 198 gcaattcaac acacagagaa atttccatac actgaaatga 40 <210> 199 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 199 aacacacaga gaatggtgga tccatacact gaaatgattg 40 <210> 200 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 200 cacagagaat ggtggatttc cactgaaatg attgttcatc 40 <210> 201 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 201 tggcatttcc aaacaccaaa acgggcacca gaatggcact t 41 <210> 202 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 202 ccaactctat taagcctggt tggttttcag agtttagcgc a 41 <210> 203 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 203 ttctgacttt ggatggagca attcaacata cagagaatgg tggattt 47 <210> 204 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 204 ctgactttgg atggagcaat tcaacataca gagaatggtg gatttccata 50 <210> 205 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 205 tgactttgga tggagcaatt caacatacag agaatggtgg atttcca 47 <210> 206 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 206 tgactttgga tggagcaatt caacatacag agaatggtgg atttccatac 50 <210> 207 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 207 gactttggat ggagcaattc aacatacaga gaatggtgga tttccat 47 <210> 208 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 208 gactttggat ggagcaattc aacatacaga gaatggtgga tttccata 48 <210> 209 <211> 49 <212> DNA <213> Nicotiana tabacum <400> 209 gactttggat ggagcaattc aacatacaga gaatggtgga tttccatac 49 <210> 210 <211> 56 <212> DNA <213> Nicotiana tabacum <400> 210 gactttggat ggagcaattc aacatacaga gaatggtgga tttccataca ctgaaa 56 <210> 211 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 211 actttggatg gagcaattca acatacagag aatggtggat ttcc 44 <210> 212 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 212 actttggatg gagcaattca acatacagag aatggtggat ttcca 45 <210> 213 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 213 actttggatg gagcaattca acatacagag aatggtggat ttccat 46 <210> 214 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 214 actttggatg gagcaattca acatacagag aatggtggat ttccata 47 <210> 215 <211> 64 <212> DNA <213> Nicotiana tabacum <400> 215 actttggatg gagcaattca acatacagag aatggtggat ttccatacac tgaaatgatt 60 gttc 64 <210> 216 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 216 ctttggatgg agcaattcaa catacagaga atggtggatt tc 42 <210> 217 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 217 cttggatgg agcaattcaa catacagaga atggtggatt tcca 44 <210> 218 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 218 ctttggatgg agcaattcaa catacagaga atggtggatt tccata 46 <210> 219 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 219 cttggatgg agcaattcaa catacagaga atggtggatt tccatac 47 <210> 220 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 220 tttggatgga gcaattcaac atacagagaa tggtggattt c 41 <210> 221 <211> 43 <212> DNA <213> Nicotiana tabacum <400> 221 tttggatgga gcaattcaac atacagagaa tggtggattt cca 43 <210> 222 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 222 tttggatgga gcaattcaac atacagagaa tggtggattt ccat 44 <210> 223 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 223 tttggatgga gcaattcaac atacagagaa tggtggattt ccata 45 <210> 224 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 224 tttggatgga gcaattcaac atacagagaa tggtggattt ccatac 46 <210> 225 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 225 tttggatgga gcaattcaac atacagagaa tggtggattt ccatacac 48 <210> 226 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 226 ttggatggag caattcaaca tacagagaat ggtggatttc ca 42 <210> 227 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 227 ttggatggag caattcaaca tacagagaat ggtggatttc cata 44 <210> 228 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 228 tggatggagc aattcaacat acagagaatg gtggatttcc at 42 <210> 229 <211> 43 <212> DNA <213> Nicotiana tabacum <400> 229 tggatggagc aattcaacat acagagaatg gtggatttcc ata 43 <210> 230 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 230 tggatggagc aattcaacat acagagaatg gtggatttcc atac 44 <210> 231 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 231 tggatggagc aattcaacat acagagaatg gtggatttcc atacac 46 <210> 232 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 232 ggatggagca attcaacata cagagaatgg tggatttcca ta 42 <210> 233 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 233 caattcaaca tacagagaat ggtggatttc catacactga a 41 <210> 234 <211> 40 <212> DNA <213> Nicotiana tabacum <400> 234 gcacttccaa acaccaaaac gggcaccaga atggcacttt 40 <210> 235 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 235 ttctgacttt ggatggagca attcaacata cagagaatgg tggattt 47 <210> 236 <211> 52 <212> DNA <213> Nicotiana tabacum <400> 236 ttctgacttt ggatggagca attcaacata cagagaatgg tggatttcca ta 52 <210> 237 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 237 ctgactttgg atggagcaat tcaacataca gagaatggtg gatttcc 47 <210> 238 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 238 ctgactttgg atggagcaat tcaacataca gagaatggtg gatttccata 50 <210> 239 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 239 tgactttgga tggagcaatt caacatacag agaatggtgg atttcca 47 <210> 240 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 240 tgactttgga tggagcaatt caacatacag agaatggtgg atttccatac 50 <210> 241 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 241 gactttggat ggagcaattc aacatacaga gaatggtgga tttccat 47 <210> 242 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 242 gactttggat ggagcaattc aacatacaga gaatggtgga tttccata 48 <210> 243 <211> 49 <212> DNA <213> Nicotiana tabacum <400> 243 gactttggat ggagcaattc aacatacaga gaatggtgga tttccatac 49 <210> 244 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 244 gactttggat ggagcaattc aacatacaga gaatggtgga tttccataca 50 <210> 245 <211> 56 <212> DNA <213> Nicotiana tabacum <400> 245 gactttggat ggagcaattc aacatacaga gaatggtgga tttccataca ctgaaa 56 <210> 246 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 246 actttggatg gagcaattca acatacagag aatggtggat tt 42 <210> 247 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 247 actttggatg gagcaattca acatacagag aatggtggat ttcc 44 <210> 248 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 248 actttggatg gagcaattca acatacagag aatggtggat ttccat 46 <210> 249 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 249 actttggatg gagcaattca acatacagag aatggtggat ttccata 47 <210> 250 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 250 actttggatg gagcaattca acatacagag aatggtggat ttccatac 48 <210> 251 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 251 actttggatg gagcaattca acatacagag aatggtggat ttccatacac 50 <210> 252 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 252 ctttggatgg agcaattcaa catacagaga atggtggatt t 41 <210> 253 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 253 ctttggatgg agcaattcaa catacagaga atggtggatt tc 42 <210> 254 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 254 cttggatgg agcaattcaa catacagaga atggtggatt tcca 44 <210> 255 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 255 ctttggatgg agcaattcaa catacagaga atggtggatt tccat 45 <210> 256 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 256 ctttggatgg agcaattcaa catacagaga atggtggatt tccatac 47 <210> 257 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 257 tttggatgga gcaattcaac atacagagaa tggtggattt cc 42 <210> 258 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 258 tttggatgga gcaattcaac atacagagaa tggtggattt ccat 44 <210> 259 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 259 tttggatgga gcaattcaac atacagagaa tggtggattt ccata 45 <210> 260 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 260 tttggatgga gcaattcaac atacagagaa tggtggattt ccatac 46 <210> 261 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 261 tttggatgga gcaattcaac atacagagaa tggtggattt ccataca 47 <210> 262 <211> 54 <212> DNA <213> Nicotiana tabacum <400> 262 tttggatgga gcaattcaac atacagagaa tggtggattt ccatacactg aaat 54 <210> 263 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 263 ttggatggag caattcaaca tacagagaat ggtggatttc c 41 <210> 264 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 264 ttggatggag caattcaaca tacagagaat ggtggatttc ca 42 <210> 265 <211> 43 <212> DNA <213> Nicotiana tabacum <400> 265 ttggatggag caattcaaca tacagagaat ggtggatttc cat 43 <210> 266 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 266 ttggatggag caattcaaca tacagagaat ggtggatttc cata 44 <210> 267 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 267 ttggatggag caattcaaca tacagagaat ggtggatttc catac 45 <210> 268 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 268 ttggatggag caattcaaca tacagagaat ggtggatttc cataca 46 <210> 269 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 269 ttggatggag caattcaaca tacagagaat ggtggatttc catacac 47 <210> 270 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 270 tggatggagc aattcaacat acagagaatg gtggatttcc at 42 <210> 271 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 271 tggatggagc aattcaacat acagagaatg gtggatttcc atac 44 <210> 272 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 272 tggatggagc aattcaacat acagagaatg gtggatttcc atacact 47 <210> 273 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 273 ggatggagca attcaacata cagagaatgg tggatttcca ta 42 <210> 274 <211> 43 <212> DNA <213> Nicotiana tabacum <400> 274 ggatggagca attcaacata cagagaatgg tggatttcca tac 43 <210> 275 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 275 gagcaattca acatacagag aatggtggat ttccatacac tg 42 <210> 276 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 276 tggcacttcc aaacaccaaa acggccacaa gaatgggact t 41 <210> 277 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 277 ccaattgtat taagcctggt tggttttcag agtttagcgc a 41 <210> 278 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 278 tgactttgga tggagcaatt caacacacag agaatggtgg atttc 45 <210> 279 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 279 tgactttgga tggagcaatt caacacacag agaatggtgg atttcca 47 <210> 280 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 280 tgactttgga tggagcaatt caacacacag agaatggtgg atttccatac 50 <210> 281 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 281 gactttggat ggagcaattc aacacacaga gaatggtgga tttccata 48 <210> 282 <211> 51 <212> DNA <213> Nicotiana tabacum <400> 282 gactttggat ggagcaattc aacacacaga gaatggtgga tttccataca c 51 <210> 283 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 283 actttggatg gagcaattca acacacagag aatggtggat tt 42 <210> 284 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 284 actttggatg gagcaattca acacacagag aatggtggat ttcc 44 <210> 285 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 285 actttggatg gagcaattca acacacagag aatggtggat ttcca 45 <210> 286 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 286 actttggatg gagcaattca acacacagag aatggtggat ttccat 46 <210> 287 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 287 actttggatg gagcaattca acacacagag aatggtggat ttccata 47 <210> 288 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 288 actttggatg gagcaattca acacacagag aatggtggat ttccatac 48 <210> 289 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 289 actttggatg gagcaattca acacacagag aatggtggat ttccatacac 50 <210> 290 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 290 ctttggatgg agcaattcaa cacacagaga atggtggatt t 41 <210> 291 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 291 ctttggatgg agcaattcaa cacacagaga atggtggatt tc 42 <210> 292 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 292 ctttggatgg agcaattcaa cacacagaga atggtggatt tcca 44 <210> 293 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 293 ctttggatgg agcaattcaa cacacagaga atggtggatt tccatac 47 <210> 294 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 294 tttggatgga gcaattcaac acacagagaa tggtggattt cc 42 <210> 295 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 295 tttggatgga gcaattcaac acacagagaa tggtggattt ccat 44 <210> 296 <211> 45 <212> DNA <213> Nicotiana tabacum <400> 296 tttggatgga gcaattcaac acacagagaa tggtggattt ccata 45 <210> 297 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 297 tttggatgga gcaattcaac acacagagaa tggtggattt ccatac 46 <210> 298 <211> 48 <212> DNA <213> Nicotiana tabacum <400> 298 tttggatgga gcaattcaac acacagagaa tggtggattt ccatacac 48 <210> 299 <211> 54 <212> DNA <213> Nicotiana tabacum <400> 299 tttggatgga gcaattcaac acacagagaa tggtggattt ccatacactg aaat 54 <210> 300 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 300 ttggatggag caattcaaca cacagagaat ggtggatttc ca 42 <210> 301 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 301 ttggatggag caattcaaca cacagagaat ggtggatttc cata 44 <210> 302 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 302 ttggatggag caattcaaca cacagagaat ggtggatttc cataca 46 <210> 303 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 303 ttggatggag caattcaaca cacagagaat ggtggatttc catacac 47 <210> 304 <211> 52 <212> DNA <213> Nicotiana tabacum <400> 304 ttggatggag caattcaaca cacagagaat ggtggatttc catacactga aa 52 <210> 305 <211> 43 <212> DNA <213> Nicotiana tabacum <400> 305 tggatggagc aattcaacac acagagaatg gtggatttcc ata 43 <210> 306 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 306 tggatggagc aattcaacac acagagaatg gtggatttcc atac 44 <210> 307 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 307 ggatggagca attcaacaca cagagaatgg tggatttcca ta 42 <210> 308 <211> 44 <212> DNA <213> Nicotiana tabacum <400> 308 ggatggagca attcaacaca cagagaatgg tggatttcca taca 44 <210> 309 <211> 49 <212> DNA <213> Nicotiana tabacum <400> 309 ggatggagca attcaacaca cagagaatgg tggatttcca tacactgaa 49 <210> 310 <211> 46 <212> DNA <213> Nicotiana tabacum <400> 310 gatggagcaa ttcaacacac agagaatggt ggatttccat acactg 46 <210> 311 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 311 tggcacttcc aaacaccaaa acggccacca gaatggcact t 41 <210> 312 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 312 ccaactctat taagcctggt tggttttcag agtttagcgc a 41 <210> 313 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 313 aacatatggg aaggttctga ctttggatgg agcaattcaa ca 42 <210> 314 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 314 atgggaaggt tctgactttg gatggagcaa ttcaacacac ag 42 <210> 315 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 315 gttctgactt tggatggagc aattcaacac acagagaatg gt 42 <210> 316 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 316 ctgactttgg atggagcaat tcaacacaca gagaatggtg gatttcc 47 <210> 317 <211> 50 <212> DNA <213> Nicotiana tabacum <400> 317 ctgactttgg atggagcaat tcaacacaca gagaatggtg gatttccata 50 <210> 318 <211> 51 <212> DNA <213> Nicotiana tabacum <400> 318 ctgactttgg atggagcaat tcaacacaca gagaatggtg gatttccata c 51 <210> 319 <211> 47 <212> DNA <213> Nicotiana tabacum <400> 319 tgactttgga tggagcaatt caacacacag agaatggtgg atttcca 47 <210> 320 <211> 41 <212> DNA <213> Nicotiana tabacum <400> 320 gactttggat ggagcaattc aacacacaga gaatggtgga t 41 <210> 321 <211> 42 <212> DNA <213> Nicotiana tabacum <400> 321 gactttggat ggagcaattc aacacacaga gaatggtgga tt 42 <210&...

Claims

1. A tobacco plant or a part thereof comprising one or more mutant alleles in at least one PMT gene selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4, wherein the tobacco plant, when grown and processed under equivalent conditions, can produce leaves containing a nicotine level lower than that of leaves from a control tobacco plant that does not have the one or more mutant alleles.

2. The tobacco plant or a part thereof according to claim 1, wherein the tobacco plant comprises one or more mutant alleles of at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

3. The tobacco plant or a part thereof according to claim 1, wherein the tobacco plant comprises one or more mutant alleles of at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

4. The tobacco plant or a part thereof according to claim 1, wherein the tobacco plant comprises one or more mutant alleles of at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

5. The tobacco plant or a part thereof according to claim 1, wherein the tobacco plant comprises one or more mutant alleles of five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4.

6. The tobacco plant or a part thereof according to any one of claims 1 to 5, wherein the tobacco plant, when grown under equivalent conditions and processed, can produce leaves containing nicotine levels lower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the nicotine levels of leaves from a control tobacco plant that does not have one or more mutant alleles.

7. The tobacco plant or a part thereof according to any one of claims 1 to 6, wherein the tobacco plant can, when grown and processed under equivalent conditions, produce leaves containing a total alkaloid level lower than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the total alkaloid level of the leaves from the control tobacco plant.

8. The tobacco plant or a part thereof, according to claim 7, which, when grown and processed under equivalent conditions, can produce leaves containing a total alkaloid level lower than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid level of leaves from the control tobacco plant.

9. The tobacco plant or a part thereof according to any one of claims 1 to 8, wherein the one or more mutant alleles comprises a mutation in a sequence region selected from the group consisting of the promoter, 5'UTR, first exon, first intron, second exon, second intron, third exon, 3'UTR, terminator, and any combination thereof.

10. The tobacco plant or a part thereof according to any one of claims 1 to 9, wherein the one or more mutant alleles include one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof.

11. The one or more mutant alleles mentioned above are as follows: PMT protein cleavage, untranslated PMT gene transcripts, non-functional PMT proteins, immature stop codons in the PMT gene, and any combination thereof. A tobacco plant or part thereof according to any one of claims 1 to 10, which provides one or more of the following:

12. The tobacco plant or a part thereof according to any one of claims 1 to 11, wherein the one or more mutant alleles comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the wild-type PMT gene.

13. The tobacco plant or a part thereof according to any one of claims 1 to 12, wherein the one or more mutant alleles include a conjugation state selected from the group consisting of homozygosity, heterozygosity, and heteroallelegenicity.

14. The tobacco plant or a part thereof according to any one of claims 1 to 12, wherein one or more mutant alleles are homozygous or heteroallelic in at least one to five PMT genes.

15. The tobacco plant or a part thereof according to any one of claims 1 to 12, wherein one or more mutant alleles are homozygous or heteroallelic in at least four PMT genes.

16. The tobacco plant or a part thereof according to any one of claims 1 to 12, wherein one or more mutant alleles are homozygous or heteroallelic in all five PMT genes.

17. The tobacco plant or a part thereof according to any one of claims 1 to 16, wherein the at least two PMT genes are PMT1a and PMT3.

18. The tobacco plant or a part thereof according to any one of the claims, wherein the tobacco plant can produce leaves containing a nicotine level selected from the group consisting of less than 0.15%, less than 0.125%, less than 0.1%, less than 0.08%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, and less than 0.01% by dry weight.

19. The tobacco plant or a part thereof according to any one of the claims, which can produce leaves containing a total alkaloid level selected from the group consisting of less than 1%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, and less than 0.2% by dry weight.

20. The tobacco plant or a part thereof according to any one of the claims, which can produce dried leaves containing a total TSNA level of 2-0.05, 1.9-0.05, 1.8-0.05, 1.7-0.05, 1.6-0.05, 1.5-0.05, 1.4-0.05, 1.3-0.05, 1.2-0.05, 1.1-0.05, 1.0-0.05, 0.9-0.05, 0.8-0.05, 0.7-0.05, 0.6-0.05, 0.5-0.05, 0.4-0.05, 0.3-0.05, 0.2-0.05, 0.15-0.05, or 0.1-0.05 ppm.

21. A group of tobacco plants according to any one of claims 1 to 20.

22. A dried tobacco material derived from a tobacco plant according to any one of claims 1 to 20.

23. The dried tobacco material according to claim 22, which is produced by a drying process selected from the group consisting of hot air drying, air drying, direct flame drying, and sun drying.

24. Contains tobacco leaves, The aforementioned tobacco leaves show reduced mold infection compared to control dried tobacco material derived from the LA Burley 21 variety. The dried tobacco material according to claim 22.

25. A tobacco blend comprising the dried tobacco material described in claim 22.

26. The tobacco blend according to claim 25, wherein the dried tobacco material constitutes at least about 10% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 35% by weight, at least 40% by weight, at least 45% by weight, at least 50% by weight, at least 55% by weight, at least 60% by weight, at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, or at least 95% by weight of the dried tobacco in the tobacco blend.

27. The tobacco blend according to claim 25, wherein the dried tobacco material constitutes at least about 10 volume%, at least 15 volume%, at least 20 volume%, at least 25 volume%, at least 30 volume%, at least 35 volume%, at least 40 volume%, at least 45 volume%, at least 50 volume%, at least 55 volume%, at least 60 volume%, at least 65 volume%, at least 70 volume%, at least 75 volume%, at least 80 volume%, at least 85 volume%, at least 90 volume%, or at least 95 volume% of the dried tobacco in the tobacco blend.

28. A tobacco product comprising the dried tobacco material described in claim 22.

29. A tobacco product according to claim 28, selected from the group consisting of cigarettes, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

30. The tobacco product according to claim 28, which is a smokeless tobacco product.

31. The tobacco product according to claim 30, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.

32. Reconstituted tobacco comprising the dried tobacco material described in claim 22.

Images