Compositions and methods based on PMT engineering for producing tobacco plants and products having altered alkaloid levels

Abstract

To provide compositions and methods relating to: tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade; development thereof via breeding or transgenic approaches; and production of tobacco products from the tobacco plants.SOLUTION: The present disclosure provides: a tobacco plant, or 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, where the tobacco plant is capable of producing a leaf having lower total alkaloid and nicotine levels than a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions; and a dried tobacco product derived from the tobacco plant.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Incorporating a sequence listing This application claims the benefit of U.S. Provisional Application No. 62 / 703,775, filed July 26, 2018, and U.S. Provisional Application No. 62 / 848,159, filed May 15, 2019, both of which are incorporated by reference in their entireties. The sequence listing contained in the file entitled "P34620WO00_SL.txt" is 200,338 bytes (measured in MS-Windows), was created on July 25, 2019, is submitted electronically herewith, and is incorporated by reference in its entirety.

[0002] Field The present disclosure provides for the genetic engineering of tobacco to modulate alkaloid and nicotine levels. [Background technology]

[0003] background Nicotine is the predominant alkaloid, typically accounting for over 90–95% of the total alkaloids in commercial tobacco cultivars. The remaining alkaloid fraction consists primarily of three additional alkaloids: nornicotine, anabasine, and anatabine. Tobacco plants with reduced nicotine levels have been obtained by modulating different nicotine biosynthetic genes and transcriptional regulators, but results have been variable and inconsistent. New technologies for reducing nicotine levels in tobacco leaves are needed. Summary of the Invention

[0004] overview The present disclosure provides tobacco plants with 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 the tobacco plants.

[0005] In one aspect, the present disclosure provides a tobacco plant or portion 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, is capable of producing leaves comprising a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant that does not have the one or more mutant alleles.

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

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

[0010] In one aspect, the present disclosure provides a tobacco plant selected from the group consisting of a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, and a quintuple mutant, as listed in Tables 8A-8E.

[0011] In one aspect, the disclosure provides a tobacco plant listed in Tables 4A-4E or Table 10. In another aspect, the disclosure provides a progeny plant of a tobacco plant in Tables 4A-4E or Table 10, derived either from self-pollination or a cross with another plant in Tables 4A-4E or Table 10.

[0012] In another aspect, the present disclosure provides tobacco plants comprising various combinations of pmt mutant alleles listed in Tables 5A-5E or Tables 12A-12E, resulting in single pmt mutants, double pmt mutants, triple mutants, quadruple mutants, or quintuple mutants. In one aspect, the present disclosure provides tobacco plants comprising the 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.

[0013] The present disclosure further provides cured tobacco, tobacco blends, and tobacco products comprising plant material derived from the disclosed tobacco plants, strains, varieties, or hybrids. [The present invention 1001] A tobacco plant or 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, is capable of producing leaves containing a nicotine level that is lower than the nicotine level in leaves from a control tobacco plant that does not have the one or more mutant alleles. [The present invention 1002] 1001. The tobacco plant or part thereof of the present invention, wherein said tobacco plant comprises one or more mutant alleles in at least two PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [The present invention 1003] 1001. The tobacco plant or part thereof of the present invention, wherein said tobacco plant comprises one or more mutant alleles in at least three PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [The present invention 1004] 1001. The tobacco plant or part thereof of the present invention, wherein said tobacco plant comprises one or more mutant alleles in at least four PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [The present invention 1005] 1001. The tobacco plant or part thereof of the present invention, wherein said tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. [The present invention 1006] 1006. The tobacco plant or part thereof of any of claims 1001 to 1005, wherein the tobacco plant, when grown and processed under equivalent conditions, is capable of producing leaves containing nicotine levels that are 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.25% lower than the nicotine level in leaves from a control tobacco plant that does not have the one or more mutant alleles. [The present invention 1007] Any of the tobacco plants or parts thereof of 1001 to 1006 of the present invention, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less 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 in leaves from the control tobacco plant when grown and processed under equivalent conditions. [The present invention 1008] 1007. A tobacco plant or part thereof of the present invention, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid level in leaves from the control tobacco plant when grown and processed under equivalent conditions. [The present invention 1009] 1008. The tobacco plant or part thereof of any of claims 1001 to 1008, wherein said one or more mutant alleles comprise a mutation in a sequence region selected from the group consisting of a promoter, a 5'UTR, a first exon, a first intron, a second exon, a second intron, a third exon, a 3'UTR, a terminator, and any combination thereof. [The present invention 1010] The tobacco plant or part thereof of any of claims 1001 to 1009 of the present invention, wherein the one or more mutant alleles comprise one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof. [The present invention 1011] The one or more mutant alleles are: PMT protein truncation, untranslated PMT gene transcript, non-functional PMT protein, premature stop codon in the PMT gene, and any combination thereof. A tobacco plant or part thereof according to any one of 1001 to 1010 of the present invention, which produces one or more of the following: [The present invention 1012] A tobacco plant or part thereof according to any one of claims 1001 to 1011, wherein the one or more mutant alleles comprise 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. [The present invention 1013] 10. The tobacco plant or part thereof of any of claims 1001 to 1012, wherein said one or more mutant alleles comprise a zygosity selected from the group consisting of homozygous, heterozygous, and heteroallelic. [The present invention 1014] 10. The tobacco plant or part thereof of any of claims 1001 to 1012, wherein said one or more mutant alleles are homozygous or heterozygous in at least one to five PMT genes. [The present invention 1015] 10. The tobacco plant or part thereof of any of claims 1001 to 1012, wherein said one or more mutant alleles are homozygous or heterozygous in at least four PMT genes. [The present invention 1016] 10. The tobacco plant or part thereof of any of claims 1001 to 1012, wherein the one or more mutant alleles are homozygous or heterozygous in all five PMT genes. [The present invention 1017] The tobacco plant or part thereof according to any one of claims 1001 to 1016, wherein the at least two PMT genes are PMT1a and PMT3. [The present invention 1018] Any of the tobacco plants or parts thereof of the present invention, wherein the tobacco plant is capable of producing 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. [The present invention 1019] Any of the tobacco plants or parts thereof of the present invention, wherein the tobacco plant is capable of producing 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. [The present invention 1020]

[0023] Any of the tobacco plants or parts thereof of the present invention, wherein the tobacco plant is capable of producing dried leaves containing total TSNA levels 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. [The present invention 1021] A population of tobacco plants according to any one of 1001 to 1020 of the present invention. [The present invention 1022] A dried tobacco material derived from any one of the tobacco plants of the present inventions 1001 to 1020. [The present invention 1023] The cured tobacco material of the present invention 1022, produced by a drying process selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. [The present invention 1024] Contains tobacco leaves, the tobacco leaves exhibit reduced mold infection compared to a control cured tobacco material from variety LA Burley 21; The dried tobacco material of the present invention 1022. [The present invention 1025] A tobacco blend comprising the dried tobacco material of the present invention. [The present invention 1026] The tobacco blend of the present invention, wherein the dried tobacco material comprises 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. [The present invention 1027] The tobacco blend of the present invention 1025, wherein the dried tobacco material comprises at least about 10% by volume, at least 15% by volume, at least 20% by volume, at least 25% by volume, at least 30% by volume, at least 35% by volume, at least 40% by volume, at least 45% by volume, at least 50% by volume, at least 55% by volume, at least 60% by volume, at least 65% by volume, at least 70% by volume, at least 75% by volume, at least 80% by volume, at least 85% by volume, at least 90% by volume, or at least 95% by volume of the dried tobacco in the tobacco blend. [The present invention 1028] A tobacco product comprising the dried tobacco material of the present invention. [The present invention 1029] 1028. The tobacco product of the present invention, 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. [The present invention 1030] The tobacco product of the present invention 1028, which is a smokeless tobacco product. [The present invention 1031] The tobacco product of the present invention 1030, wherein the smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. [The present invention 1032] A reconstituted tobacco comprising the dried tobacco material of the present invention.

[0014] A brief description of arrays SEQ ID NOs: 1-5 set forth exemplary genomic sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4, respectively, from the TN90 reference genome.

[0015] SEQ ID NOs: 6-10 set forth exemplary cDNA sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4 from TN90, respectively.

[0016] SEQ ID NOs: 11-15 set forth exemplary polypeptide sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4, respectively, from TN90.

[0017] SEQ ID NOs: 16 to 22 show exemplary guide RNA sequences.

[0018] SEQ ID NOs: 23-200, 410-441, 474-505, 538-569, 602-633, and 666-697 set forth exemplary edited pmt mutant sequences. [Brief explanation of the drawings]

[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 in the NLM(Ph Ph) background), a PMT RNAi transgenic strain in the VA359 background, and the low-nicotine KY171 ("LN KY171") cultivar (a KY171 background harboring a nic1 and nic2 double mutation) compared with their respective normal-alkaloid control strains: NLM(Ph Ph), VA359, and KY171 backgrounds. [Figure 3] Total alkaloid levels in various low-alkaloid strains: CS15, PMT RNAi, and LN KY171, compared to their respective normal-alkaloid control strains: NLM (Ph Ph), VA359, and KY171 backgrounds. [Figure 4] Leaf yield in various low-alkaloid lines: CS15, PMT RNAi, and LN KY171, compared to their respective normal-alkaloid control lines: NLM (Ph Ph), VA359, and KY171 backgrounds. [Figure 5] Leaf quality in various low-alkaloid strains: CS15, PMT RNAi, and LN KY171, compared to their respective normal-alkaloid control strains: NLM (Ph Ph), VA359, and KY171 backgrounds. [Figure 6A]Photographs showing mold growth on cured tobacco containing 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 legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7] Depiction of fungal infection observed in the strains investigated in Figures 6A-6E. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description Unless otherwise defined, the 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 can be used to implement the present disclosure. In fact, the present disclosure is in no way limited to the methods and materials described. For the purposes of this disclosure, the following terms are defined below.

[0021] Any references cited herein, including, for example, all patents and publications, are incorporated by reference in their entirety.

[0022] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise clear from the context. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0023] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical value by 10%.

[0024] As used herein, words such as "less than," "more than," "at least," "at most," "approximately," "below," "greater than," and "about," when used in conjunction with a series of numerical values, modify each value in the series. For example, the phrase "less than 1%, 2%, or 3%" is equivalent to "less than 1%, less than 2%, or less than 3%."

[0025] As used herein, tobacco plants refer to plants derived from 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-adenosyl-methionine as a cofactor. This is the step in which precursor metabolites are involved in nicotine biosynthesis. PMT enzymes are classified as EC 2.1.1.53 under the enzyme classification system. In Nicotiana tabacum, five genes encode putrescine N-methyltransferases, designated PMT1a, PMT1b, PMT2, PMT3, and PMT4. Table 1A lists the genomic DNA, cDNA, and protein sequences of these five PMT genes in TN90 plants. This disclosure describes compositions and methods used to edit PMT genes to produce pmt mutant plants with reduced nicotine levels while maintaining leaf quality.

[0027] As used herein, "PMT1b" or "PMT1b gene" refers to a genetic locus in tobacco that encodes a polypeptide having the exemplary amino acid sequence of TN90 set forth in SEQ ID NO:11.

[0028] As used herein, "PMT1a" or "PMT1a gene" refers to a genetic locus in tobacco that encodes a polypeptide having the exemplary amino acid sequence of TN90 set forth in SEQ ID NO:12.

[0029] As used herein, "PMT2" or "PMT2 gene" refers to a genetic locus in tobacco that encodes a polypeptide having the exemplary amino acid sequence of TN90 set forth in SEQ ID NO:13.

[0030] As used herein, "PMT3" or "PMT3 gene" refers to a genetic locus in tobacco that encodes a polypeptide having the exemplary amino acid sequence of TN90 set forth in SEQ ID NO:14.

[0031] As used herein, "PMT4" or "PMT4 gene" refers to a genetic locus in tobacco that encodes a polypeptide having the exemplary amino acid sequence of TN90 set forth in SEQ 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 the product encoded by the gene. Such a modification can be in any sequence region of the gene, such as the promoter, 5'UTR, exon, intron, 3'UTR, or terminator region. In one aspect, 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 genetic locus in which the allelic locus contains a mutation.

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

[0034] As used herein, gene or locus names are capitalized and italicized, e.g., PMTi a, PMTi b, PMTi 2, PMTi 3, and PMTi 4. Protein or polypeptide names are capitalized and not italicized, e.g., PMTi a, PMTi b, PMTi 2, PMTi 3, and PMTi 4. Variant names (to refer to common mutations in a gene or group of genes or to refer to specific mutant alleles) are lowercase and italicized, e.g., pmt, pmtla, pmtlb, pmt2, pmt3, and pmt4.

[0035] In one aspect, the present disclosure provides a tobacco plant or portion 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, is capable of producing leaves comprising nicotine levels lower than the nicotine levels of leaves from a control tobacco plant lacking the one or more mutant alleles. In one aspect, a single pmt mutant tobacco plant is provided. In another aspect, the single pmt mutant tobacco plant, when grown under similar growth conditions, comprises nicotine at a level less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant lacking the single pmt mutation. In further embodiments, the single pmt mutant tobacco plants, when grown under similar growth conditions, contain nicotine at a level of 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant not having the single pmt mutant.

[0036] In another embodiment, the tobacco plant comprises 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 a level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant not having the double pmt mutation. In further embodiments, the double pmt mutant tobacco plants contain nicotine at levels between 1% and 5%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 95% of the nicotine level of a control plant not having the double pmt mutation when grown under similar growth conditions.

[0037] In a further aspect, the tobacco plant comprises 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 aspect, a triple pmt mutant tobacco plant is provided. In another aspect, the triple pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at a level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant without the triple pmt mutation. In further embodiments, the triple pmt mutant tobacco plants contain nicotine at levels that are 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have the triple pmt mutation when grown under similar growth conditions.

[0038] In another embodiment, the tobacco plant comprises 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 a level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have the quadruple pmt mutation. In further embodiments, the quadruple pmt mutant tobacco plants contain nicotine at levels that are 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have the quadruple pmt mutation when grown under similar growth conditions.

[0039] In a further aspect, the tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMTla, PMTlb, PMT2, PMT3, and PMT4. In one aspect, a quintuple pmt mutant tobacco plant is provided. In another aspect, the quintuple pmt mutant tobacco plant, when grown under similar growth conditions, contains nicotine at a level that is less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nicotine level of a control plant that does not have the quintuple pmt mutation. In further embodiments, the quintuple pmt mutant tobacco plants, when grown under similar growth conditions, contain nicotine at a level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the nicotine level of a control plant that does not have 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 listed in Tables 8A-8E. In another embodiment, the tobacco plant comprises one or more pmt mutant alleles listed in Tables 5A-5E and 12A-12E. Each and every combination of pmt mutant alleles listed in Tables 5A-5E and 12A-12E is also provided to result in a single pmt mutant, double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant. Each mutant locus can be either homozygous or heterozygous, or can comprise heteroallelic combinations. In another embodiment, the tobacco plant comprises a combination of pmt mutant genotypes as shown for each individual line listed in Tables 4A-4E and 10. In one aspect, the tobacco plant comprises 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 aspect, the disclosure provides a double pmt mutant, triple mutant, quadruple mutant, or quintuple mutant comprising 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, a tobacco plant, when grown and processed under equivalent conditions, can produce leaves containing nicotine levels that are less 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 level of leaves from a control tobacco plant. In another embodiment, a tobacco plant, when grown and processed under equivalent conditions, can produce leaves containing nicotine levels that are between 1% and 5%, 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 95% of the nicotine level of a control tobacco plant.

[0042] In another embodiment, the tobacco plants, when grown and processed under equivalent conditions, are capable of producing leaves containing a total alkaloid level that is less 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 leaves from control tobacco plants. In another embodiment, the tobacco plants, when grown and processed under equivalent conditions, are capable of producing leaves containing a total alkaloid level that is 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 95% of the total alkaloid level of control tobacco plants.

[0043] In further embodiments, the tobacco plants are capable of producing leaves containing total alkaloid levels that are less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid levels in leaves from control tobacco plants when grown and processed under equivalent conditions.

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

[0046] In one embodiment, the mutant pmt allele results in one or more of a PMT protein truncation, an untranslatable PMT gene transcript, a non-functional PMT protein, a premature 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 comprises a zygosity selected from the group consisting of homozygous, heterozygous, and heteroallelic. In another embodiment, the pmt mutant is homozygous or heteroallelic in at least one, two, three, four, or five PMT genes. In one embodiment, the pmt mutant is homozygous or heteroallelic in at least four PMT genes. In another embodiment, the pmt mutant is homozygous or heteroallelic in all five PMT genes. In another embodiment, the pmt mutant comprises a mutation in PMT1a and PMT3.

[0049] In one aspect, the tobacco plant is capable of producing 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.

[0050] In another aspect, the tobacco plant is capable of producing 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.

[0051] In further embodiments, the tobacco plants are capable of producing dried leaves containing total TSNA levels 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.

[0052] In one embodiment, the tobacco plant, when dried, is capable of producing leaves having a USDA grade index value selected from the group consisting of 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. In another embodiment, the tobacco plant, when dried, is capable of producing leaves having a USDA grade index value equivalent to that of a control plant when grown and dried under similar conditions, the control plant sharing essentially the same genetic background as the tobacco plant except for the modification. In a further embodiment, the tobacco plant, when dried, is capable of producing leaves having a USDA grade index value 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 value of the control plant when grown under similar conditions, the control plant sharing essentially the same genetic background as the tobacco plant except for the modification. In further embodiments, the tobacco plants, when dried, are capable of producing leaves having a USDA grade index value that is 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA grade index value of the control plant. In further embodiments, the tobacco plants, when dried, are capable of producing leaves having a USDA grade index value that is 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the USDA grade index value of the control plant.

[0053] In one aspect, the tobacco plant contains nicotine at a level that is 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, and the control plant shares essentially the same genetic background as the tobacco plant except for the modification.

[0054] In a further aspect, 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 a MATE transporter.

[0055] In one embodiment, the tobacco plant comprises one or more pmt mutant alleles and further comprises a mutation in an ERF gene at the Nic2 locus. In one embodiment, the tobacco plant further comprises 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 one embodiment, the tobacco plant further comprises one or more mutations in ERF189, ERF115, or both.

[0056] In one embodiment, the tobacco plant comprises one or more qpt mutant alleles and further comprises a mutation in an ERF gene at the Nic1 locus (or Nic1b locus, as found in PCT / US2019 / 013345, filed January 11, 2019, published as WO / 2019 / 140297). See also WO / 2018 / 237107. In one embodiment, the tobacco plant further comprises 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 aspect, the present disclosure further provides a pmt mutant tobacco plant or portion thereof comprising 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 cured, is capable of producing leaves having a USDA grade index value of 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater. In another embodiment, such pmt mutant tobacco plants are capable of producing leaves containing nicotine levels of less than 0.02% and, when dried, having a USDA Grade Index value of 70 or greater. In a further embodiment, such tobacco plants are capable of producing leaves containing nicotine levels of less than 0.01% and, when dried, having a USDA Grade Index value of 70 or greater.

[0058] In one aspect, the disclosure also provides a tobacco plant or portion thereof comprising a non-transgenic mutation, wherein the non-transgenic mutation reduces the nicotine level or total alkaloid level of the tobacco plant to less than 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the nicotine level of a control plant when grown under similar growth conditions, wherein the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value equivalent to the USDA grade index value of the control plant, and wherein the control plant shares essentially the same genetic background as the tobacco plant except for the non-transgenic mutation.

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

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

[0061] Unless otherwise specified, nicotine or alkaloid levels (or another leaf chemistry or characteristic) of tobacco plants are measured after topping in pooled leaf samples collected from leaf numbers 3, 4, and 5 after topping. As used herein, whenever a comparison between leaves from two plants (e.g., a mutant plant and a control plant) is referred to, leaves from the same or equivalent leaf position(s) and developmental stage(s) are intended, and the comparison can demonstrate effects due to genotypic differences rather than other factors. Illustratively, 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 to a control tobacco plant of the same tobacco variety.

[0062] Nicotine or alkaloid levels (or other leaf chemistry or characteristic) of tobacco plants can also be measured by alternative methods. In one embodiment, the nicotine or alkaloid levels (or other leaf chemistry or characteristic) of tobacco plants are measured after flower removal in the leaves with the highest levels of nicotine or alkaloids (or other leaf chemistry or characteristic). In one embodiment, the nicotine or alkaloid levels of tobacco plants are measured after flower removal in 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 leaves. In another embodiment, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plants are measured after flower thinning in two or more pools of consecutive leaf numbers selected from the group consisting of 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, and 30. In another embodiment, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plants are measured after flower thinning in leaves of leaf numbers selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30. In another embodiment, nicotine or alkaloid levels (or another leaf chemistry or characteristic) of tobacco plants are measured after flower removal in pools of two or more leaves having a number of leaves selected from the group consisting of 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30. In another embodiment, nicotine or alkaloid levels (or another leaf chemistry or characteristic) of tobacco plants are measured after flower removal in pools of three or more leaves having a number of leaves selected from the group 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 leaf's position on the tobacco stem, with leaf number 1 being the youngest leaf (top) after pruning and the highest leaf number being assigned to the oldest leaf (bottom).

[0064] The population of tobacco plants or collection of tobacco leaves for determining an average measurement (e.g., alkaloid or nicotine level or leaf grading) can be of any size, e.g., 5, 10, 15, 20, 25, 30, 35, 40, or 50. Industry-accepted standard protocols are followed for determining the average measurement or grading index value.

[0065] As used herein, "deflowering" refers to removing the shoot apex, including the shoot apex containing the shoot apical meristem (SAM), the flower, and up to several adjacent leaves, when tobacco plants are nearing plant maturity and at the beginning of reproductive growth. Typically, tobacco plants are deflowered at the button stage (shortly after flowers begin to appear). For example, tobacco plants grown in greenhouses or fields can be deflowered when 50% of the plants have at least one flowering. Deflowering tobacco plants eliminates apical dominance and induces increased alkaloid production.

[0066] Unless otherwise indicated, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plants are measured two weeks after flower thinning. Alternatively, other time points can be used. In one embodiment, the nicotine or alkaloid levels (or another leaf chemistry or characteristic) of the tobacco plants are measured about 1, 2, 3, 4, or 5 weeks after flower thinning. In another embodiment, the nicotine, alkaloid, or polyamine levels (or another leaf chemistry or characteristic) of the tobacco plants are measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after flower thinning.

[0067] As used herein, "similar growing conditions" or "equivalent growing conditions" refer to similar environmental conditions and / or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices contribute to or 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). Agronomic practices include, for example, seeding, clipping, undercutting, transplanting, topping, and suckering. See Chapters 4B and 4C in 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 commercial cigarette tobacco, accounting for approximately 90% of the alkaloid content in Nicotiana tabacum. Other major alkaloids in tobacco include cotinine, nornicotine, myosmine, nicotyrine, anabasine, and anatabine. Minor 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 gas-liquid chromatography, high-performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay-based quantification. For example, nicotinic alkaloid levels can be measured by GC-FID in accordance with CORESTA Recommended Method No. 7, 1987 and ISO standards (ISO TC 126N 394 E). For a method using gas-liquid chromatography equipped with a capillary column and an FID detector, see Hibi et al., Plant Physiology 100:826-35 (1992).

[0070] Unless otherwise noted, alkaloid and nicotine levels are measured using methods according to CORESTA Method No. 62, "Determination of Nicotine in Cigarettes and Tobacco Products by Gas Chromatography," February 2005, and the Centers for Disease Control and Prevention protocol for the analysis of nicotine, total moisture, and pH in smokeless tobacco products, published in the Federal Register, Vol. 64, No. 55, March 23, 1999 (and amended, Vol. 74, No. 4, January 7, 2009). Alternatively, total alkaloids in tobacco can be measured using a segmented-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). Briefly, tobacco samples can be dried, crushed, and extracted prior to analysis of total alkaloids and reducing sugars. The method then uses acetic acid / methanol / water extraction and charcoal for decolorization. Total alkaloid determination is based on the reaction of nicotine alkaloids with cyanogen chloride in the presence of aromatic amines to form a colored complex that is measured at 460 nm. Unless otherwise specified, total alkaloid or nicotine levels presented herein are on a dry weight basis (e.g., percent total alkaloids or percent nicotine).

[0071] In one embodiment, the tobacco plant comprises, on a dry weight basis, about 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%, 2% or more of the above-mentioned compounds. %, 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 tobacco plant comprises, on a dry weight basis, about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5, 1.5% to 1.6%, 1.6% to 1.8%, 1.8% to 1.9%, 1.9% to 2.0%, 2.0% to 2.1%, 2.0% to 2.2%, 2.0% to 2.3%, 2.1% to 2.4%, 2.2% to 2.5%, 2.3% to 2.6%, 2.4% to 2.7%, 2.5% to 2.8%, 2.6% to 2.9%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3.0%, 2.0% to 3.1%, 2.1% to 3.2%, 2.2% to 3.3%, 2.3% to 3.4%, 2.4% to 2.5%, 2.5% to 3.6%, 2.6% to 3.7%, 2.7% to 3.8%, 2.8 and an average nicotine level or total alkaloid level selected from the group consisting of 0.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2%, 2% to 2.1%, 2.1% to 2.2%, 2.2% to 2.3%, 2.3% to 2.4%, 2.4% to 2.5%, 2.5% to 2.6%, 2.6% to 2.7%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3%, 3% to 3.1%, 3.1% to 3.2%, 3.2% to 3.3%, 3.3% to 3.4%, 3.4% to 3.5%, and 3.5% to 3.6%. In further embodiments, the tobacco plants comprise an average nicotine level or total alkaloid level selected from the group consisting of about 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.

[0072] The present disclosure also provides tobacco plants with altered nicotine levels that do not adversely affect other tobacco traits, such as leaf grade index values. In one aspect, low-nicotine or nicotine-free tobacco varieties provide commercially acceptable grades of cured tobacco. Tobacco grade is assessed based on factors including, but not limited to, leaf position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to leaf strength and color depth and brilliance), hygroscopicity (the ability of tobacco leaves to absorb and retain ambient moisture), and green hue or color cast. Leaf grade can be determined, for example, using official standard grades published by the U.S. Department of Agriculture's Agricultural Marketing Service (7 U.S.C. § 511).For example, the Official Standard Grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93) (55 F.R. 40645), effective November 5, 1990; the Official Standard Grades for Flue-Cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective March 27, 1989 (54 F.R. 7925); the Official Standard Grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective January 8, 1965 (29 F.R. 16854); the Official Standard Grades for Ohio Cigar-Leaf Tobacco (U.S. Type 41), effective December 8, 1963 (29 F.R. 16854); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969; Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55) (34 F.R. 17061), effective November 20, 1969; Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), effective April 1971. USDA grade index values ​​can be determined according to industry-recognized grade indexes. See, e.g., 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 foregoing references are incorporated by reference in their entirety).In one embodiment, the USDA Grade Index is a numerical representation of the federal grade from 0 to 100, which is a weighted average of all leaf positions. The higher the Grade Index, the higher the quality. Alternatively, leaf grade can be determined via hyperspectral imaging. See, e.g., WO 2011 / 027315 (published March 10, 2011, and incorporated by reference in its entirety).

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

[0074] As used herein, tobacco aroma compounds are compounds related to the flavor and aroma of tobacco smoke.These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cembrenoids and labdenoids, diterpenes, and sugar esters.The concentration of tobacco aroma 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 sugar(s)" refers to any sugar (monosaccharide or polysaccharide) that has a free or potentially free aldehyde or ketone group. 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 modifying the sensory impact of nicotine and other tobacco alkaloids. An 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. Reduction of sugar levels can be measured using a segmented-flow colorimetric method developed for the analysis of tobacco samples, adapted by Skalar Instrument Co. (West Chester, PA) and described in Davis, Tobacco Science 20:139-144 (1976). For example, the sample is dialyzed against a sodium carbonate solution. Copper neocuproine is added to the sample, and the solution is heated. The neocuproine copper chelate is reduced in the presence of sugars to give a colored complex that is 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, which reduce or eliminate 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), or UV irradiation, X-rays, 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 ed), 1987), transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and 5,013,658), and T-DNA insertion techniques (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists of chemically induced random point mutations across the length of the genome. Rapid neutron mutagenesis involves exposing seeds to neutron irradiation, which causes large deletions through double-stranded DNA breaks. Transposon tagging involves inserting a transposon into an endogenous gene to reduce or eliminate gene expression. Types of mutations that can exist in tobacco genes include, for example, point mutations, deletions, insertions, duplications, and inversions. Desirably, such mutations exist in the coding region of a tobacco gene, but mutations in promoter regions, introns, or untranslated regions of a tobacco gene may also be desirable.

[0078] In addition, a rapid and automatable method for screening for chemically induced mutations, TILLING (targeted local disruption of the genome), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the present disclosure. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457. Mutations that affect gene expression or disrupt 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 can be particularly effective in disrupting protein function. In one embodiment, the tobacco plant contains a nonsense (e.g., stop codon) mutation in one or more of the PMT genes described herein.

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

[0080] In one aspect, the present disclosure also provides tobacco strains with altered nicotine levels while maintaining commercially acceptable leaf quality. These strains can be produced by introducing mutations into one or more PMT genes via precise genome engineering techniques, such as transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / Csm1 systems, and combinations thereof (see, e.g., U.S. Patent Application Publication No. 2017 / 0233756). See, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013).

[0081] Screening and selection of mutagenized tobacco plants can be performed by any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detecting polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, next-generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzymatic or ribozyme activity of polypeptides and polynucleotides, as well as protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme-linked immunoassays for detecting 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. Methods for performing the cited techniques are all known.

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

[0083] As used herein, "editing" or "genome editing" refers to 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 genomic nucleic acid sequence, or removal or replacement of an endogenous plant genomic nucleic acid sequence. In one aspect, the edited nucleic acid sequence provided 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 to the endogenous nucleic acid sequence. In one embodiment, the edited nucleic acid sequences provided have 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 to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-10 and fragments thereof. In another embodiment, the edited nucleic acid sequences provided have 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 to 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-stranded DNA breaks at target sites in genome sequences, which are then repaired by the natural process of homologous recombination (HR) or non-homologous end joining (NHEJ). Sequence modifications then occur at the break site, which can include deletions or insertions resulting in gene disruption in the case of NHEJ, or the integration of donor nucleic acid sequences by HR. In one embodiment, a provided method includes editing a plant genome using a provided nuclease, and 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 through HR with a donor polynucleotide. In one embodiment, the provided mutations are caused by genome editing using a nuclease. In another embodiment, the provided mutations are caused by non-homologous end joining or homologous recombination.

[0085] Meganucleases, commonly identified in microorganisms, are unique enzymes with high activity and long recognition sequences (>14 bp) that result in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (e.g., 14-40 bp). Engineering meganucleases can be more challenging than engineering 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 have been used to create novel meganuclease variants that recognize unique sequences and have improved nuclease activity.

[0086] ZFNs are synthetic proteins consisting of engineered zinc finger DNA-binding domains fused to the cleavage domain of the FokI restriction endonuclease. Due to the modification of the zinc finger DNA-binding domain, ZFNs can be designed to cleave double-stranded DNA of almost any length. ZFNs form dimers from monomers consisting of the non-specific DNA cleavage domain of the FokI endonuclease fused to an engineered zinc finger array that binds to the target DNA sequence.

[0087] The DNA-binding domain of a ZFN typically consists of an array of three to four zinc fingers. The amino acids at positions -1, +2, +3, and +6 relative to the beginning of the zinc finger ∞-helix, which contribute to site-specific binding to the target DNA, can be altered and customized to accommodate specific target sequences. Other amino acids form a consensus backbone to generate 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, thus requiring two ZFNs with their C-terminal regions to bind to opposite DNA strands (separated by 5-7 bp) from the cleavage site. A ZFN monomer can cleave a target site if the two ZF binding sites are palindromic. 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 engineered to function 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 re-engineered using one of a variety of methods, so customized ZFNs can theoretically be constructed to target almost any gene sequence. Commonly available methods for engineering zinc finger domains include context-dependent assembly (CoDA), oligomerized pool engineering (OPEN), and modular assembly.

[0090] TALEN is an artificial restriction enzyme that is generated by fusing a transcription activator-like effector (TALE) DNA binding domain with a FokI nuclease domain. When each member of a TALEN pair binds to the DNA site adjacent to the target site, the FokI monomer dimerizes and causes double-stranded DNA breaks at the target site. As used herein, the term TALEN has a broad meaning and includes monomeric TALENs that can cut double-stranded 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 function together to cut DNA at the same site.

[0091] Transcription activator-like effectors (TALEs) can be engineered to bind virtually any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens in 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 33-34 amino acid monomers. The amino acids in each monomer are highly conserved except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat variable dimers (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVD preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and the RVD can regulate consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allowed the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0092] In addition to the wild-type FokI cleavage domain, mutant FokI cleavage domain variants have been designed to improve cleavage specificity and activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains oriented and spaced appropriately to target genomic sites. 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 allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. 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 system, CRISPR / Csm1, or CRISPR / Cpf1 system is an alternative to the FokI-based methods ZFN and TALEN. The CRISPR system is based on RNA-guided engineered nucleases that use 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 system of bacteria and archaea, protecting them from invading nucleic acids such as viruses by cleaving foreign DNA in a sequence-dependent manner. Immunity is achieved through the incorporation of a short segment of invading DNA, known as a spacer, between two adjacent repeats at the proximal end of the CRISPR locus. During subsequent encounters with the invading DNA, the spacer-containing CRISPR array is transcribed and processed into approximately 40-nt-long small interfering CRISPR RNA (crRNA), which, in combination with trans-activating CRISPR RNA (tracrRNA), activates and guides the Cas9 nuclease, which cleaves the homologous double-stranded DNA sequence known as the protospacer 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, NAG. Specificity is provided by a so-called "seed sequence" approximately 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 and Csm1 act in a manner similar to Cas9, but Cpf1 and Csm1 do not require tracrRNA.

[0096] In yet another embodiment, the tobacco plants provided herein comprise one or more pmt mutations, and further comprise one or more mutations that confer a reduced amount of nornicotine at one or more loci encoding nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) compared to a control plant that lacks one or more mutations at one or more loci encoding nicotine demethylase (see U.S. Patent Nos. 8,319,011, 8,124,851, 9,187,759, 9,228,194, 9,228,195, and 9,247,706). In one embodiment, the tobacco plants described further comprise 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 comprises a mutation that enables it to produce leaves containing anabasine levels that are less than the anabasine levels of leaves from a wild-type control tobacco plant when grown and processed under equivalent conditions. In another embodiment, the pmt mutant tobacco plant further comprises a mutation that enables it to produce leaves containing anabasine levels that are less than 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the anabasine levels of leaves from a wild-type control tobacco plant when grown and processed under equivalent conditions.

[0098] In one embodiment, the pmt mutant tobacco plant contains an additional mutation that, when grown and processed under equivalent conditions, can produce leaves containing anatabine levels that are reduced by at least twofold compared to leaves from a control tobacco plant. In another embodiment, the pmt mutant tobacco plant contains an additional mutation that, when grown and processed under equivalent conditions, can produce leaves containing anatabine levels that are reduced by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 13-fold compared to leaves from a wild-type control tobacco plant. In one embodiment, the mutation that provides lower levels of anatabine is a mutation described in U.S. Patent Application Publication Nos. 2014 / 0283165 and 2016 / 0010103. In another embodiment, the pmt mutant further contains a mutation in a quinolate phosphoribosyltransferase (QPT) or quinolinate synthase (QS) gene. In a further embodiment, the pmt mutant plant further contains a transgene or mutation that suppresses the expression or activity of the QPT or QS gene.

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

[0100] In one aspect, pmt mutant tobacco plants are capable of producing cured 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 are capable of producing dried leaves containing total NNN levels of between 2 and 0.05, 1.9 and 0.05, 1.8 and 0.05, 1.7 and 0.05, 1.6 and 0.05, 1.5 and 0.05, 1.4 and 0.05, 1.3 and 0.05, 1.2 and 0.05, 1.1 and 0.05, 1.0 and 0.05, 0.9 and 0.05, 0.8 and 0.05, 0.7 and 0.05, 0.6 and 0.05, 0.5 and 0.05, 0.4 and 0.05, 0.3 and 0.05, 0.2 and 0.05, 0.15 and 0.05, or 0.1 and 0.05 parts per million (ppm).

[0102] In one aspect, pmt mutant tobacco plants are capable of producing cured leaves containing total nicotine-derived nitrosamine ketone (NNK) levels 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 are capable of producing dried leaves containing total NNK levels 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.

[0104] In one embodiment, the pmt mutant tobacco plant further comprises a mutation or transgene that provides increased levels of one or more antioxidants. In another embodiment, the pmt mutant tobacco plant further comprises a genetic modification in an endogenous gene, further comprising increased levels of one or more antioxidants in its cured leaves compared to control cured tobacco leaves lacking the genetic modification, the endogenous gene encoding an antioxidant biosynthetic enzyme, an antioxidant regulatory transcription factor, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In a further embodiment, the pmt mutant tobacco plant further comprises a transgene, further comprising increased levels of one or more antioxidants in its cured leaves compared to control cured tobacco leaves lacking the transgene, the transgene encoding or directly regulating an antioxidant biosynthetic enzyme, 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, chorismate mutase, and arogenate dehydrotase (ADT). In another embodiment, the pmt mutant tobacco plant further comprises one or more transgenes or genetic modifications to increase antioxidants or decrease one or more TSNAs, as described in WIPO Publication No. 2018 / 067985 or U.S. Publication No. 2018 / 0119163.

[0105] In one aspect, the tobacco plants described are modified tobacco plants. As used herein, "modified" in the context of plants refers to plants that contain genetic alterations that have been introduced for a particular purpose and beyond natural polymorphisms.

[0106] In one embodiment, the tobacco plant described is a cisgenic plant. As used herein, "cisgenesis" or "cisgenic" refers to the genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoters, donor nucleic acids, selection genes) have only plant origin (i.e., no non-plant-origin components are used). In one embodiment, the provided plant, plant cell, or plant genome is cisgenic. The provided cisgenic plants, plant cells, and plant genomes can lead to ready-to-use tobacco strains. In another embodiment, the provided tobacco plants do not contain non-tobacco genetic material or sequences.

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

[0108] In one aspect, 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), phosphoribosylanthranilate isomerase (PRAI), quinolinate phosphoribosyltransferase (QPT), and S-adenosylmethionine synthase (SAMS). Two candidate genes (A622 and NBBl) have been proposed for nicotine synthase, which catalyzes the condensation step between nicotinic acid derivatives and methylpyrrolinium cations, but the mechanism remains unclear. See US2007 / 0240728A1 and US2008 / 0120737A1. A622 encodes an isoflavone reductase-like protein. In addition, several transporters may be involved in nicotine permeation. A transporter gene named MATE has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)).

[0109] In one embodiment, provided tobacco plants comprise one or more pmt mutations and further comprise reduced levels of mRNA, protein, or both of one or more genes encoding products selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl, relative to a control tobacco plant. In another embodiment, provided tobacco plants comprise one or more pmt mutations and further comprise a transgene that directly suppresses expression of one or more genes encoding products selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl. In another embodiment, provided tobacco plants comprise one or more pmt mutations and further comprise a transgene or mutation that suppresses expression or activity of one or more genes encoding products selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBBl.

[0110] In one aspect, the provided tobacco plants are derived from a tobacco variety selected from the group consisting of flue-cured, air-cured, dark air-cured, dark flue-cured, Galpao, and Oriental tobacco. In another aspect, the provided tobacco plants are derived from a tobacco variety selected from the group consisting of Burley, Maryland, and dark tobacco.

[0111] In one embodiment, the provided tobacco plant is of the flue-cured tobacco background or exhibits one or more flue-cured tobacco characteristics described herein. Flue-cured tobacco (also known as Virginia tobacco or light tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is often referred to as "light tobacco" because it turns golden to deep orange during curing. Flue-cured tobacco has a light, distinctive aroma and taste. Flue-cured tobacco is generally high in sugar and low in oil. The major growing countries for flue-cured tobacco are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, provided low alkaloid or low nicotine tobacco plants or seeds are in a flue-cured tobacco background 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 foregoing varieties.In another aspect, the provided low alkaloid or low nicotine tobacco plants or seeds are selected from the group consisting of 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, Reams In a further embodiment, the low-alkaloid or low-nicotine tobacco plant, seed, hybrid, variety, or strain is in a flue-cured tobacco background selected from the group consisting of K326, K346, and NC196.

[0112] In one embodiment, the provided tobacco plant is in the context of air-cured tobacco or exhibits one or more characteristics of air-cured tobacco described herein. Air-cured tobaccos include burley, Maryland, and dark tobacco. A common factor is that they are cured primarily without artificial heat and humidity sources. Burley tobacco is light to dark brown in color, high in oil, and low in sugar. Burley tobacco is air-cured in sheds. Major burley growing countries 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 Maryland growing countries include the United States and Italy. In one aspect, provided low alkaloid or low nicotine tobacco plants or seeds are in a burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky14, Ky907, Ky910, Ky8959, 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 aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or strain is on any burley background selected from the group consisting of TN90, KT209, KT206, KT212, and HB 4488. In another aspect, the provided low alkaloid or low nicotine tobacco plant or seed is on a Maryland tobacco background selected from the group consisting of Md10, Md40, Md201, Md609, Md872, and Md341.

[0113] In one aspect, the provided tobacco plants are on a dark air-cured tobacco background or exhibit one or more characteristics of dark air-cured tobacco described herein. Dark air-cured tobacco is distinguished from other varieties primarily by its curing process, which gives dark air-cured tobacco its medium-to-dark brown color and distinctive aroma. Dark air-cured tobacco is primarily used for chewing tobacco and snuff. In one aspect, the low-alkaloid or low-nicotine tobacco plants or seeds are provided on a dark air-cured tobacco background 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 provided tobacco plant is in the background of dark flame-cured tobacco or exhibits one or more characteristics of dark flame-cured tobacco described herein. Dark flame-cured tobacco is generally cured on the floor of a closed curing shed over a low-burning fire. The leaves have a low sugar content but a high nicotine content. Dark flame-cured tobacco is used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-flavored cigars. The main growing areas of dark flame-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one embodiment, the provided low alkaloid or low nicotine tobacco plants or seeds are in a dark flue-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT508, DT518, DT592, KY171, DF911, DF485, TND94, TND950, VA309, and VA359.

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

[0116] In one aspect, the low alkaloid or low nicotine tobacco plant, seed, hybrid, variety, or strain is selected from the group consisting of 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 0, TN97, TN97LC, TND94, TND950, TR (Tom Rosson) Madore, VA309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, Shirey LC, or any commercially available tobacco variety by standard tobacco breeding techniques known in the art.

[0117] Any specific varieties of the aforementioned dark air-cured, burley, Maryland, dark flame-cured, or Oriental varieties are listed for illustrative purposes only, and any additional dark air-cured, burley, Maryland, dark flame-cured, or Oriental varieties are also contemplated in this application.

[0118] Also provided are populations of the described tobacco plants. In one embodiment, the population of tobacco plants has a plant density of about 5,000 to about 8,000, about 5,000 to about 7,600, about 5,000 to about 7,200, about 5,000 to about 6,800, about 5,000 to about 6,400, about 5,000 to about 6,000, about 5,000 to about 5,600, about 5,000 to about 5,200, about 5,200 to about 8,000, about 5,600 to about 8,000, about 6,000 to about 8,000, about 6,400 to about 8,000, about 6,800 to about 8,000, about 7,200 to about 8,000, or about 7,600 to about 8,000 plants per acre. In another embodiment, the population of tobacco plants is in a soil type having low to medium fertility.

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

[0120] Cured tobacco materials made from the described low-alkaloid or low-nicotine tobacco plants are also provided. Additionally, cured tobacco materials made from the described tobacco plants with higher levels of total alkaloids or nicotine are provided.

[0121] "Curing" is a retrogradation process that reduces moisture and results in the breakdown of chlorophyll, turning tobacco leaves golden brown and converting starch to sugars. Thus, cured tobacco has a higher reducing sugar content and a lower starch content compared to harvested green leaves. In one aspect, the provided green-leaf tobacco can be dried using conventional means, such as hot air curing, barn drying, open flame curing, air drying, or sun drying. For a description of different types of curing methods, see, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured tobacco is typically aged in compressed conditions in wooden drums (e.g., hogsheads) or cardboard boxes for several years (e.g., 2-5 years), resulting in a moisture content ranging from 10% to about 25%. See U.S. Pat. Nos. 4,516,590 and 5,372,149. The dried and aged tobacco can then be further processed. Further processing includes the introduction of steam at various temperatures, pasteurization, and conditioning the tobacco under vacuum with or without fermentation. Fermentation is typically characterized by an initial high moisture content, the generation of heat, and a 10-20% loss in dry weight. See, e.g., U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149, U.S. Publication No. 2005 / 0178398, and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, and 4,987,907. In one embodiment, the cured tobacco material of the present disclosure is sun-dried. In another embodiment, the cured tobacco material of the present disclosure is flue-dried, air-dried, or flame-dried.

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

[0123] Tobacco plants, cultivars, 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 mold infection compared to the low-alkaloid tobacco cultivar LA Burley 21 (LA BU21). Similarly, tobacco plants, cultivars, and strains provided herein that include RNAi constructs that down-regulate 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 mold infection compared to the low-alkaloid tobacco cultivar LA Burley 21 (LA BU21).

[0124] LA BU21 is a low-total alkaloid tobacco strain produced by incorporating low-alkaloid gene(s) from Cuban cigar varieties into Burley 21 through multiple 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) in its parent, Barley 21. LA BU21 has a leaf grade well below commercially acceptable standards.

[0125] In one embodiment, cured tobacco leaves comprising mutant alleles of pmt1a are free of observable mold infection. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt1b are free of observable mold infection. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt2 are free of observable mold infection. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt3 are free of observable mold infection. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt4 are free of observable mold infection. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt1a, pmt1b, pmt2, pmt3, and pmt4 are free of observable mold infection.

[0126] In one embodiment, cured tobacco leaves comprising mutant alleles of pmt1a comprise reduced mold infection compared to control cured tobacco leaves from various LA BU21 strains. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt1b comprise reduced mold infection compared to control cured tobacco leaves from various LA BU21 strains. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt2 comprise reduced mold infection compared to control cured tobacco leaves from various LA BU21 strains. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt3 comprise reduced mold infection compared to control cured tobacco leaves from various LA BU21 strains. In another embodiment, cured tobacco leaves comprising mutant alleles of pmt4 comprise reduced mold infection compared to control cured tobacco leaves from various LA BU21 strains. In another aspect, cured tobacco leaves comprising mutant alleles of pmt1a, pmt1b, pmt2, pmt3, and pmt4 comprise reduced mold infection compared to control cured tobacco leaves derived from cultivar LA BU21.

[0127] In one embodiment, cured leaves from a tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14 contain no observable mold infection. In another embodiment, cured leaves from a tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14 contain reduced mold infection compared to control cured tobacco leaves from variety LA BU21.

[0128] In one embodiment, cured leaves from a tobacco plant, variety, or strain comprising one or more pmt mutations provided in any one of Tables 5A-5E and 12A-12E are free of observable mold infection. In another embodiment, cured leaves from a tobacco plant, variety, or strain comprising one or more pmt mutations provided in any one of Tables 5A-5E and 12A-12E are free of observable mold infection compared to control cured leaves from variety LA BU21.

[0129] In one embodiment, cured leaves from a tobacco plant, variety, or line comprising a mutant allele of pmt1a comprise a higher leaf grade than control cured leaves from variety LA BU21. In one embodiment, cured leaves from a tobacco plant, variety, or line comprising a mutant allele of pmt1b comprise a higher leaf grade than control cured leaves from variety LA BU21. In one embodiment, cured leaves from a tobacco plant, variety, or line comprising a mutant allele of pmt2 comprise a higher leaf grade than control cured leaves from variety LA BU21. In one embodiment, cured leaves from a tobacco plant, variety, or line comprising a mutant allele of pmt3 comprise a higher leaf grade than control cured leaves from variety LA BU21. In one embodiment, cured leaves from a tobacco plant, variety, or line comprising a mutant allele of pmt4 comprise a higher leaf grade than control cured leaves from variety LA BU21. In another aspect, cured tobacco leaves from a plant, variety, or strain comprising a mutant allele of pmt1a, a mutant allele of pmt1b, a mutant allele of pmt2, a mutant allele of pmt3, and a mutant allele of pmt4 comprise a higher leaf grade than control cured leaves of LA BU21.

[0130] In one embodiment, cured leaves from a tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14 comprise a higher leaf grade than control cured leaves from variety LA BU21.

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

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

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

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

[0135] In one embodiment, the mold that infects the cured tobacco is a mold of a genus selected from the group consisting of Cladosporium, Penicillium, Alternaria, Aspergillus, and Mucor.

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

[0137] Tobacco products provided include, but are not limited to, cigarette products (e.g., cigarettes and bidis), cigar products (e.g., cigars 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, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Publication No. US2006 / 0191548.

[0138] As used herein, a "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 filtration material), tipping paper that holds the cigarette paper (including the filler) to the filter, and any adhesives that hold these components together. "Filler" includes (1) all tobacco, including but not limited to reconstituted tobacco and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials, and other spices that may accompany tobacco wrapped in cigarette paper), (3) casing, (4) flavorings, and (5) all other additives (mixed with the tobacco and substitutes and wrapped within the cigarette).

[0139] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap materials, processed into sheet form, and cut into strips to resemble tobacco. In addition to cost savings, reconstituted tobacco is of great importance because it contributes to the taste of a cigarette through the process of flavor development using a reaction between ammonia and sugars.

[0140] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been processed through expansion with a suitable gas so that the tobacco is "filled," reducing its density and increasing its fill capacity, thereby reducing the weight of the tobacco used in the cigarette.

[0141] The plant-derived tobacco products of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles containing filter elements, where the rod of smokable material comprises dried tobacco within a tobacco blend. In one aspect, the tobacco product of the present disclosure is 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 aspect, the tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not combusted and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snuff, and dry snuff. Chewing tobacco is roughly divided tobacco leaves, typically packaged in large pouches for use in plugs or twists. Moist smokeless tobacco is moist, finely divided tobacco provided in loose or pouch form, typically packaged in a round can and used by adult tobacco consumers as pinches or pouches between the cheek and gum. Snuff is heat-treated smokeless tobacco. Dry snuff is finely ground tobacco used in the mouth or nose. In a further aspect, the tobacco product of the present disclosure is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an electronic cigarette, and an electronic evaporation device.

[0142] In one embodiment, the tobacco product of the present disclosure can be a blended tobacco product. In another embodiment, the tobacco product of the present disclosure can be a low-nicotine tobacco product. In a further embodiment, the tobacco product of the present disclosure can include nornicotine at a level of less than about 3 mg / g. For example, the nornicotine content in such products can 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 dried tobacco material or tobacco product has, on a dry weight basis, about 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%, 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 dried tobacco material or tobacco product has, on a dry weight basis, about 0.01% to 0.02%, 0.02% to 0.05%, 0.05% to 0.75%, 0.75% to 0.1%, 0.1% to 0.15%, 0.15% to 0.2%, 0.2% to 0.3%, 0.3% to 0.35%, 0.35% to 0.4%, 0.4% to 0.5%, 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1%, 1% to 1.1%, 1.1% to 1.2%, 1.2% to 1.3%, 1.3% to 1.4%, 1.4% to 1.5%, or 1.5% to 2.5% of the total dry tobacco material or tobacco product. %, 1.5% to 1.6%, 1.6% to 1.7%, 1.7% to 1.8%, 1.8% to 1.9%, 1.9% to 2%, 2% to 2.1%, 2.1% to 2.2%, 2.2% to 2.3%, 2.3% to 2.4%, 2.4% to 2.5%, 2.5% to 2.6%, 2.6% to 2.7%, 2.7% to 2.8%, 2.8% to 2.9%, 2.9% to 3%, 3% to 3.1%, 3.1% to 3.2%, 3.2% to 3.3%, 3.3% to 3.4%, 3.4% to 3.5%, and 3.5% to 3.6%. In further embodiments, the provided dried tobacco material or tobacco product comprises an average nicotine level or total alkaloid level selected from the group consisting of about 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] The present 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. To introgress or breed desirable traits or alleles into tobacco plants, DNA fingerprinting, SNP mapping, haplotype mapping, or similar techniques may be used in marker-assisted selection (MAS) breeding programs. For example, breeders can use F1 hybrid plants to create segregating populations in the F2 or backcross generation, or further cross F1 hybrid plants with other donor plants possessing agronomically desirable genotypes. Plants in the F2 or backcross generation can be screened for desirable agronomic traits or desirable chemical properties using techniques known in the art or one of the techniques listed herein. Depending on the expected inheritance pattern or the MAS technique used, self-pollination of selected plants can be performed before each backcross cycle to aid in the identification of desirable 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 can be a flue-cured variety, a burley variety, a dark air-cured variety, a dark flame-cured variety, or an Oriental variety. Other breeding techniques can be found, for example, in Wernsman, E.A., and Rufty, R.C. 1987 Chapter Seventeen. Tobacco. Pages 669-698 Cultivar Development. Crop Species. W.H. Fehr (ed.), MacMillan Publishing Company, Inc., New York, NY (incorporated herein by reference in their entireties).

[0145] The results of plant breeding programs using the described tobacco plants include useful strains, cultivars, varieties, progeny, inbred lines, and hybrids of the present disclosure. As used herein, the term "variety" refers to a group of plants that share certain characteristics that separate them from other plants of the same species. Varieties are often, but not always, sold commercially. While a variety possesses one or more characteristic traits, it is further characterized by very little overall variation among individuals within the variety. "Pure line" varieties can 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 variety can be essentially derived from another strain or variety. As defined by the International Convention for the Protection of New Varieties of Plants (December 2, 1961, November 10, 1972, October 23, 1978, and March 19, 1991, revised at Geneva), a variety is "essentially derived" from an original variety if it: a) is derived primarily from, or is 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) is clearly distinguishable from the original variety; and c) matches 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 acts of derivation. Essentially derived varieties can be obtained, for example, by selection of natural or induced mutants, somaclonal variants, variant individuals derived from plants of the original variety, backcrossing, or 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 "strain," as distinguished from a variety, most often refers to a group of plants used non-commercially, for example, in botanical research. Strains typically show little overall variation among individuals for one or more traits of interest, although there may be some variation among individuals for other traits.

[0146] In one aspect, the present disclosure provides a tobacco plant, variety, line, or cell comprising one or more pmt mutations provided in any one of Tables 5A-5E and 12A-12E.

[0147] In another aspect, the present disclosure provides a tobacco plant, variety, strain, or cell derived from any tobacco plant, variety, or strain provided in any one of Tables 4A-4E, Table 10, or Table 14.

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

[0149] In one aspect, the present disclosure provides methods for introgressing a low-nicotine trait into a tobacco variety, the methods comprising: (a) crossing a first tobacco variety including the low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; (b) genotyping the one or more progeny tobacco plants for a pmt mutant allele selected from those listed in Tables 4A-4E, 5A-5E, 10, and 12A-12E; and (c) selecting a progeny tobacco plant that includes a pmt mutant allele. In another aspect, the methods further comprise backcrossing the selected progeny tobacco plant with the second tobacco variety. In a further aspect, the methods further comprise: (d) crossing the selected progeny plant with itself or the second tobacco variety to produce one or more additional progeny tobacco plants; and (e) selecting an additional progeny tobacco plant that includes the low-nicotine trait. In one aspect, the selecting in step (e) comprises marker-assisted selection. In one aspect, these methods produce single gene conversions comprising a low-nicotine trait. In one aspect, these methods produce single gene conversions comprising a pmt mutant allele. In one aspect, the second tobacco variety is an elite variety. In another aspect, the genotyping step of these methods involves one or more molecular marker assays. In another aspect, genotyping may comprise polymorphic markers comprising polymorphisms selected from the group consisting of single nucleotide polymorphisms (SNPs), insertions or deletions (indels) in DNA sequences, simple sequence repeats (SSRs) of DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs.

[0150] As used herein, a "locus" refers to a chromosomal location 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, an "allele" refers to an alternative nucleic acid sequence at a gene or a particular locus (e.g., a nucleic acid sequence at a gene or locus that is different from other alleles for the same gene or locus). Such alleles can be considered (i) wild-type, or (ii) mutant if 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 can have reduced or eliminated gene activity or expression levels compared to the wild-type allele. For diploid organisms such as tobacco, a first allele can occur on one chromosome, and a second allele can occur 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 homologous chromosome of the plant 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.Plants that are homozygous for mutant alleles at a locus can contain the same mutant allele or different mutant alleles when they are heteroallelic or biallelic.

[0151] As used herein, "transgenesis" or "transgenic" refers to the transfer of a desired allele at one genetic locus to another genetic locus from one genetic background.

[0152] As used herein, "crossed" or "crossing" means producing offspring through fertilization (e.g., of a cell, seed, or plant), 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 progeny plant back to one of its parents. In a backcrossing scheme, the "donor" parent refers to the parent plant that has the desired gene or locus to be introduced. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the 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 the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on. In one aspect, backcrossing is performed repeatedly, with the progeny individuals of each successive backcross generation being themselves backcrossed to the same parental genotype.

[0154] As used herein, "single-gene transformed" or "single-gene transformation" refers to a plant developed using a plant breeding technique known as backcrossing or through genetic engineering, in which a single gene is transferred into a variety through backcrossing techniques or through genetic engineering, and essentially all of the desired morphological and physiological characteristics of the variety are restored.

[0155] As used herein, "elite variety" means any variety that has resulted from breeding and selection for superior agronomic performance.

[0156] As used herein, "selecting" or "selection" in the context of marker-assisted selection or breeding refers to the act of picking or choosing desired individuals, usually from a population, based on certain predetermined criteria.

[0157] As used herein, the term "trait" refers to one or more detectable characteristics of a cell or organism that can be influenced by genotype. A phenotype can be observable with the naked eye or by any other means of evaluation known in the art, such as microscopy, biochemical analysis, genomic analysis, assays for specific disease resistance, etc. In some cases, a phenotype is directly controlled by a single gene or locus, e.g., a "monogenic trait." In other cases, a phenotype is due to several genes.

[0158] As used herein, "marker assay" means a method of detecting a polymorphism at a particular genetic locus using a particular method, such as measurement of at least one phenotype (such as seed color, flower color, or other visually detectable trait), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), random 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 the process of selecting a desired trait(s) in a plant(s) by detecting one or more nucleic acids from the plant, linking those nucleic acids to the desired trait(s), and then selecting plants or germplasm that have those one or more nucleic acids.

[0160] As used herein, "polymorphism" refers to the presence of one or more variations in a population. Polymorphisms can manifest as variations in the nucleotide sequence of a nucleic acid or variations in the amino acid sequence of a protein. Polymorphisms include the presence of one or more variations in the nucleic acid sequence or nucleic acid characteristics of one or more loci in a population of one or more individuals. Variations can include, but are not limited to, base changes of one or more nucleotides, insertions of one or more nucleotides, or deletions of one or more nucleotides. Polymorphisms 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 meiotic process (e.g., unequal crossing over, genome duplication, and chromosome breakage and fusion). Variations can be commonly found or present at low frequencies within a population, with the former having greater utility in general plant breeding, and the latter being associated with rare but important phenotypic variations. Useful polymorphisms can include single nucleotide polymorphisms (SNPs), insertions or deletions (Indels) in DNA sequences, simple sequence repeats (SSRs) in DNA sequences, restriction fragment length polymorphisms (RFLPs), and tag SNPs.In addition, genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5' untranslated regions of genes, 3' untranslated regions of genes, microRNAs, siRNAs, resistance loci, satellite markers, transgenes, mRNAs, dsmRNAs, transcriptional profiles, and methylation patterns can also comprise polymorphisms.In addition, the presence, absence, or variation of the copy number of the above can also comprise polymorphisms.

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

[0162] As used herein, the terms "marker" or "molecular marker" or "marker locus" refer to a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. Any detectable polymorphic trait can be used as a marker, as long as it is differentially inherited and exhibits linkage disequilibrium with the phenotypic trait of interest. Thus, each marker is an index of a specific segment of DNA with a unique nucleotide sequence. Map positions provide a measure of the relative location of specific markers relative to one another. When a trait is described as associated with a given marker, it will be understood that the actual DNA segment whose sequence influences the trait generally cosegregates with the marker. More precise and reliable localization of the trait can be obtained when markers are identified on both sides of the trait. Relatively simple molecular tests can detect the presence of a trait by measuring the appearance of a marker(s) in the progeny of a cross, without actually assessing the appearance of the trait itself (which can be difficult and time-consuming because it requires growing plants at a stage and / or under environmental conditions in which the trait can be expressed).

[0163] It is understood that any tobacco plant of the present disclosure can further comprise additional agronomically desirable traits, for example, by transformation with a genetic construct or transgene using techniques known in the art. Examples of desirable traits include, but are not limited to, herbicide tolerance, pest resistance, disease resistance, high yield, high grade index, dryness, drying quality, machine harvestability, holding capacity, leaf quality, height, plant maturity (e.g., early maturity, early-to-mid maturity, mid-to-late maturity, or late maturity), stem size (e.g., small, medium, or large stem), or number of leaves per plant (e.g., low (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or high (16-21 leaves)), or any combination. In one embodiment, the disclosed low-nicotine or nicotine-free tobacco plants or seeds contain one or more transgenes expressing one or more insecticidal proteins, e.g., Bacillus thuringiensis crystal protein, or a plant insecticidal protein from Bacillus cereus, such as VIP3 (see, e.g., Estruch et al. (1997) Nat. Biotechnol. 15:137). In another embodiment, the tobacco plants further contain a transgenic trait that confers resistance to brown stem rot (U.S. Pat. No. 5,689,035) or cyst nematode (U.S. Pat. No. 5,491,081).

[0164] The present disclosure also provides pmt mutant tobacco plants that contain altered nicotine or total alkaloid levels but have yields equivalent to those of the corresponding original tobacco plants without such altered nicotine levels. In one embodiment, the pmt mutants provide yields selected from the group consisting of about 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 per acre. In another embodiment, the pmt mutant tobacco variety provides a yield selected from the group consisting of about 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 per acre. In further embodiments, pmt mutant tobacco plants provide a yield that is 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the yield of a control plant having essentially the same genetic background, except for the pmt mutation(s). In further embodiments, pmt mutant tobacco plants provide a yield that is 70% to 125%, 75% to 120%, 80% to 115%, 85% to 110%, or 90% to 100% of the yield of a control plant having essentially the same genetic background, except for the pmt mutation(s).

[0165] In one aspect, the disclosed tobacco plants (e.g., low-nicotine, nicotine-free, or low-alkaloid tobacco varieties) include modifications that confer a desirable trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) without substantially affecting a trait selected from the group consisting of yield, maturity and senescence, susceptibility to insect feeding damage, polyamine content after flower thinning, chlorophyll level, mesophyll cell number per unit leaf area, and end-product quality after curing.

[0166] In one aspect, the disclosed tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, nicotine-free, or low alkaloid) and further comprise traits substantially equivalent to an unmodified control plant, the traits being selected from the group consisting of yield, maturity and senescence, susceptibility to insect feeding damage, polyamine content after flower thinning, chlorophyll level, mesophyll cell number per unit leaf area, and end-product quality after drying.

[0167] In one embodiment, the disclosed tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprise a yield that is greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 100%, greater than 105%, greater than 110%, greater than 115%, greater than 120%, greater than 125%, greater than 130%, greater than 135%, or greater than 140% relative to the yield of an unmodified control plant. In one embodiment, the disclosed tobacco plants comprise a modification that confers a desired trait (e.g., low nicotine, nicotine-free, or low alkaloids) and further comprise a yield that is between 70% and 140%, 75% and 135%, 80% and 130%, 85% and 125%, 90% and 120%, 95% and 115%, or 100% and 110% relative to the yield of an unmodified control plant. In one aspect, the disclosed tobacco plants include a modification that confers a desired trait (e.g., low nicotine, nicotine-free, or low alkaloid) and further include a yield that is 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, 90% to 100%, 95% to 105%, 105% to 115%, 110% to 120%, 115% to 125%, 120% to 130%, 125 to 135%, or 130% to 140% relative to the yield of an unmodified control plant.

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

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

[0170] The plants can be used to form single-cross tobacco F1 hybrids. Pollen from a male parent plant is manually transferred to an emasculated or male-sterile female parent plant to form F1 seeds. Alternatively, a three-way cross can be performed in which a single-cross F1 hybrid is used as the female parent and crossed with a different male parent. As another alternative, the F1 progeny of two different single crosses can be crossed with themselves to create a double-cross hybrid. When forming double-cross hybrids, self-incompatibility is particularly advantageous, as it can prevent self-pollination of the female parent.

[0171] In one embodiment, the low-nicotine or nicotine-free tobacco variety is male sterile. In another embodiment, the low-nicotine or nicotine-free tobacco variety is cytoplasmic male sterile. Male sterile tobacco plants can be produced by any method known in the art. Methods for producing male sterile tobacco are described in Wernsman, EA, and Rufty, RC 1987 Chapter Seventeen. Tobacco. Pages 669-698 Cultivar Development. Crop Species. W.H. Fehr (ed.), MacMillan Publishing Company, Inc., New York, NY 761 pp.

[0172] In one aspect, the present disclosure provides a male sterile tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of Tables 5A-5E and 12A-12E.

[0173] In another aspect, the present disclosure provides a male sterile tobacco plant, variety, or line derived from any tobacco plant, variety, or line provided in any one of Tables 4A-4E, Table 10, or Table 14.

[0174] In one aspect, the present disclosure provides male sterile line dCS11. In another aspect, the present disclosure provides male sterile line dCS12. In another aspect, the present disclosure provides male sterile line dCS13. In another aspect, the present disclosure provides male sterile line dCS14. In another aspect, the present disclosure provides male sterile line dCS15. In another aspect, the present disclosure provides male sterile line dCS16. In another aspect, the present disclosure provides male sterile line dCS17. In another aspect, the present disclosure provides male sterile line dCS18. In another aspect, the present disclosure provides male sterile line dS697.

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

[0176] The cells, tissues, and organs may be derived from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, pods, flowers, inflorescences, stalks, pedicels, styles, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, or vascular tissue. In another aspect, the present disclosure provides chloroplasts of tobacco plants. In a further aspect, the present disclosure provides epidermal cells, stomatal cells, leaves or root hairs, storage roots, or tubers. In another aspect, the present 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 aspect, the present disclosure provides tobacco endosperm. In another aspect, the present disclosure provides tobacco endosperm cells. In a further aspect, the present disclosure provides male or female sterile tobacco plants that are unable to reproduce without human intervention.

[0178] In one aspect, the disclosure provides a nucleic acid molecule comprising 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 to a sequence selected from the group consisting of SEQ ID NOs: 1-10, and fragments thereof. In one aspect, the disclosure provides a polypeptide or protein comprising 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-15.

[0179] As used herein, the term "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the same residues in the two sequences when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity are used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution.

[0180] The present disclosure further provides methods for producing tobacco products comprising tobacco material from the disclosed tobacco plants. In one embodiment, the method includes conditioning aged tobacco material produced from the tobacco plants to increase its moisture content from about 12.5% ​​to about 13.5% to about 21% and blending the conditioned tobacco material to produce a desired blend. In one embodiment, the method for producing a tobacco product further includes casing or flavoring the blend. Typically, during the casing process, casing or source materials are added to the blend to balance its chemical composition and enhance its quality and develop 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] The provided tobacco material can also be processed using methods including, but not limited to, heat treatment (e.g., cooking, toasting), flavoring, enzyme treatment, expansion, and / or curing. Both fermented and non-fermented tobaccos can be processed using these techniques. Examples of suitable processed tobaccos include dark air-cured, dark flame-cured, burley, hot-air-cured, and cigar filler or wrapper products, as well as products from whole leaf stemming operations. In one embodiment, the tobacco fiber comprises up to 70% dark tobacco on a green weight basis. For example, the tobacco can be conditioned by heating, sweetening, and / or pasteurization steps, as described in U.S. Publication Nos. 2004 / 0118422 or 2005 / 0178398.

[0182] The provided tobacco material can be fermented. Fermentation is typically characterized by an initial high moisture content, the generation of heat, and a 10-20% loss in dry weight. See, e.g., U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, and 5,372,149. Fermentation can alter the aroma of the leaves and can also change either or both of their color and texture. During the fermentation process, gases can be released, oxygen can be taken up, pH can change, and the amount of moisture retained can change. See, e.g., U.S. Publication No. 2005 / 0178398 and Tso (1999, Chapter 1 of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). The dried or dried and fermented tobacco may be further processed (e.g., cut, expanded, blended, milled, or ground) before being incorporated into an oral product. The tobacco is optionally long-cut, fermented, dried, moist tobacco having an oven volatiles content of 48-50% by weight before being mixed with the copolymer, and optionally flavorants and other additives.

[0183] In one embodiment, the provided tobacco material can be processed to a desired size. In one embodiment, the tobacco fiber can be processed to have an average fiber size of less than 200 micrometers. In one embodiment, the tobacco fiber is 75 to 125 micrometers. In another embodiment, the tobacco fiber is processed to have a size of 75 micrometers or less. In one embodiment, the tobacco fiber includes long cut tobacco, which 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 inch to about 1 inch. The double cut tobacco fiber can have a variety of particle sizes, with about 70% of the double cut tobacco fiber having a mesh size of -20 mesh to 80 mesh.

[0184] The provided tobacco material can be processed to have a total oven volatile content of about 10% by weight or greater, about 20% by weight or greater, about 40% by weight or greater, 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 volatiles" is determined by calculating the percentage weight loss of a sample after drying the sample in a preheated forced draft oven at 110°C for 3.25 hours. Oral products can have a total oven volatile content that differs from the oven volatile content of the tobacco fiber used to make the oral product. The processing steps described can reduce or increase oven volatile content.

[0185] Having now generally described the present disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure, unless otherwise specified. [Example]

[0186] Example 1: Expression profiling of five PMT genes Nicotine biosynthesis begins with the conversion of the polyamine putrescine to N-methylputrescine by the enzyme putrescine N-methyltransferase (PMT). This is the step in which precursor metabolites are involved in nicotine biosynthesis. Genes encoding PMTs (PMT1a, PMT1b, PMT2, PMT3, and PMT4) are present in the tobacco (Nicotiana tabacum) genome. Table 1A lists the genomic DNA, cDNA, and protein sequences of five PMT genes. Tables 1B and 1C provide sequence identities among the five PMT genes. Pooled expression levels from preflowering to harvest provide support, without being limited to any particular theory, that PMT1a and PMT3 represent the two major PMT genes (Figure 1).

[0187] (Table 1A) Sequences of five tobacco PMT genes TIFF2025061515000002.tif51128

[0188] (Table 1B) cDNA sequence identity among five tobacco PMT genes determined by Clustal2.1 TIFF2025061515000003.tif34128

[0189] (Table 1C) Protein sequence identity among five tobacco PMT genes determined by Clustal2.1 TIFF2025061515000004.tif34128

[0190] Table 1D. Annotation of PMT1b genome sequence (SEQ ID NO: 1). TIFF2025061515000005.tif80128

[0191] Table 1E: Annotation of the PMT1b genome sequence (SEQ ID NO: 2) TIFF2025061515000006.tif80128

[0192] Table 1F. Annotation of PMT2 genomic sequence (SEQ ID NO: 3) TIFF2025061515000007.tif80128

[0193] Table 1G. Annotation of PMT3 genome sequence (SEQ ID NO: 4) TIFF2025061515000008.tif80128

[0194] Table 1H. Annotation of PMT4 genome sequence (SEQ ID NO: 5). TIFF2025061515000009.tif80128

[0195] Example 2: PMT genome editing and tobacco strain development PMT knockout mutants are generated by editing various PMT genes. Tobacco protoplasts are transfected using polyethylene glycol (PEG) with a plasmid encoding the genome editing technology 1 (GET1) protein or genome editing technology (GET) 2 protein and a specific guide RNA (gRNA) targeting the PMT gene at the desired location. Table 2 lists the gRNA sequences used for PMT editing. Some gRNAs (e.g., numbers 6 and 7) are pooled together to target multiple PMT genes in a single transfection.

[0196] The transfected protoplasts are then immobilized on 1% agarose beads and subjected to tissue culture. Once the calli have grown to approximately 1 mm in diameter, they are plated onto TOM2 plates. The calli are screened for insertions or deletions (indels) at the target location using fragment analysis. Candidates showing a size shift compared to the wild-type control are selected for further culture, and the resulting shoots are again tested by fragment analysis to confirm the presence of indels. Rooted shoots are potted and sequenced at the target location to determine the exact deleted sequence. Young leaves from each plant are harvested and PCR-amplified for PMT fragments using the phirekit. The PMT library for each line is indexed, and 384 lines are pooled and sequenced using Miseq.

[0197] SNP analysis was performed to determine both the sequence of the precise edited pmt mutant allele and the zygosity status at each PMT locus. Table 3 provides zygosity information for representative edited plants. Tables 4A-4E provide sequence information for indels in each edited line of various tobacco varieties (e.g., K326, TN90, NLM, Oriental). Tables 5A-5E provide approximately 40 nucleotides of genomic sequence from each pmt mutant allele with the edited site in the center of the genomic sequence (e.g., 20 nucleotides on each side of the sequence site of the deletion or insertion).

[0198] (Table 2) gRNA sequences and their target genes used in the two genome editing techniques. "Y" indicates that the gRNA targets that PMT gene, while "-" indicates that the gRNA does not target that PMT gene. TIFF2025061515000010.tif94156

[0199] Table 3. Zygosity of individual PMT loci in selected pmt mutants in various backgrounds generated by genome editing using GET2. Number 1 represents homozygosity for a single mutant allele. Numbers 2-5 represent heterozygous allele combinations with 2-5 indels. A hyphen indicates no data. Detailed genotype information is shown in Tables 4A-4D. TIFF2025061515000011.tif187136TIFF2025061515000012.tif226136TIFF2025061515000013.tif226136TIFF2025061515000014.tif61136

[0200] (Table 4A) Mutant pmt alleles in 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 biallelic mutations in PMT1b. One of the two alleles has a four-nucleotide deletion corresponding to nucleotides 416-419 of the PMT1b cDNA sequence. The other allele has a two-nucleotide deletion corresponding to nucleotides 418-419 of the PMT1b cDNA sequence. For sequences longer than 10 nucleotides, SEQ ID NOs have been assigned and are shown. TIFF2025061515000015.tif217113TIFF2025061515000016.tif217153TIFF2025061515000017.tif21765

[0201] (Table 4B) Mutant pmt alleles of TN90 generated by genome editing using GET2 TIFF2025061515000018.tif233141TIFF2025061515000019.tif233150TIFF2025061515 000020.tif233149TIFF2025061515000021.tif233153TIFF2025061515000022.tif23369

[0202] (Table 4C) Mutant pmt alleles of NLMz generated by genome editing using GET2. NLMz refers to the narrow-leaf Madol cultivar, which contains three loss-of-function mutations in three nicotine demethylase genes (CYP82E4, CYP82E5v2, and CYP82E10). TIFF2025061515000023.tif230133TIFF2025061515000024.tif230150TIFF2025061515000025.tif230154TIFF2025061515000026.tif23083

[0203] Table 4D. Oriental tobacco mutant pmt alleles generated by genome editing using GET2 TIFF2025061515000027.tif219140TIFF2025061515000028.tif219152TIFF2025061515000029.tif21959

[0204] Table 4E. Mutant pmt alleles of NLM(Ph Ph) tobacco generated by genome editing using GET1. TIFF2025061515000030.tif23632

[0205] (Table 5A) List of exemplary mutant alleles obtained in the PMT1b gene. The sequences of the mutant alleles listed herein and in Tables 5B-5E represent approximately 40 nucleotides of genomic sequence from each edited PMT gene with the editing site in the center of the genomic sequence (e.g., 20 nucleotides on each side of the sequence site of the deletion or insertion). These mutant alleles correspond to those listed in Tables 4A-4E. TIFF2025061515000031.tif196119TIFF2025061515000032.tif196145TIFF2025061515000033.tif196154TIFF2025061515000034.tif19635

[0206] (Table 5B) List of exemplary mutant alleles obtained in the PMT1a gene TIFF2025061515000035.tif188145TIFF2025061515000036.tif188154TIFF2025061515000037.tif188137

[0207] Table 5C: List of exemplary mutant alleles obtained in the PMT2 gene TIFF2025061515000038.tif173143TIFF2025061515000039.tif173154TIFF2025061515000040.tif173154

[0208] Table 5D. List of exemplary mutant alleles obtained in the PMT3 gene TIFF2025061515000041.tif196145TIFF2025061515000042.tif196154TIFF2025061515000043.tif196119

[0209] Table 5E: List of exemplary mutant alleles obtained in the PMT4 gene TIFF2025061515000044.tif202145TIFF2025061515000045.tif202145

[0210] Example 3: Alkaloid analysis of PMT-edited strains Genome-edited and control tobacco plants were grown in 10-inch pots in a greenhouse using 75 ppm fertilizer. At the flowering stage, plants were deflowered. Two weeks after deflowering, leaf blade samples from the third, fourth, and fifth leaves were collected and alkaloid levels were measured (see Tables 6A-6C). Measurements were performed according to CORESTA Method 62, "Determination of Nicotine in Cigarettes and Tobacco Products by Gas Chromatography Analysis" (February 2005), and the Centers for Disease Control and Prevention Protocol for the Analysis of Nicotine, Total Moisture, and pH in Smokeless Tobacco Products, published in the Federal Register, Vol. 64, No. 55, March 23, 1999 (amended Vol. 74, No. 4, January 7, 2009).

[0211] Briefly, 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 percent (wt%) either as is or on a dry weight basis. To report data on a dry weight basis, oven volatiles (OV) determination is required. Unless otherwise specified, total or individual alkaloid or nicotine levels presented herein are on a dry weight basis (e.g., percent total alkaloids or percent nicotine).

[0212] Plants will also be planted in the field, harvested, and tested for alkaloid and TSNA levels in the cured tobacco. Both leaf yield and leaf grade will also be evaluated for PMT-edited plants. Additionally, different mutant combinations of individual PMT genes will be generated and tested (e.g., single, double, triple, or quadruple mutants).

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

[0214] Example 5: Obtaining tobacco lines with edited mutant alleles in one or more PMT genes Tobacco lines bearing mutations in individual PMT genes or selected combinations of PMT genes can be derived from the tobacco lines listed in Table 3. Quintuple, quadruple, triple, or double mutants (having mutations in five, four, three, or two PMT genes, respectively) can be crossed with non-mutated control lines and progeny plants segregating for specific PMT mutation combinations can be selected. Tables 7A-7E represent possible mutant combinations. Each mutant gene can be either homozygous or heterozygous for the mutation. Each of the mutant alleles listed in Tables 4A-4E and Table 10 can be used to generate single, double, triple, quintuple, or quadruple mutants. Exemplary individual pmt mutant alleles are listed in Tables 9A-9E.

[0215] Example 6: Further reduction of total alkaloids by combining pmt mutations 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 quinolinate 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 quinuple pmt mutants). The resulting qpt / pmt or qs / pmt mutant combinations are then tested for alkaloid and TSNA levels in cured tobacco. Both leaf yield and leaf grade are also evaluated.

[0216] (Table 6A) Alkaloid levels in PMT-edited lines of K326 (shown here and in Tables 6B, 6C, and 7 as percentages of dry weight per gram of leaf blade). TIFF2025061515000046.tif104128

[0217] Table 6B. Alkaloid levels in PMT-edited strains of TN90 TIFF2025061515000047.tif188128

[0218] Table 6C. Alkaloid levels in PMT-edited strains of narrow-leaf madol (NLM). TIFF2025061515000048.tif131128

[0219] Table 7: Relative changes in nicotine and total alkaloid levels in quintuple pmt knockout mutants across various cultivars. The average percent levels of nicotine and total alkaloids are calculated based on percent level data from individual strains, as shown in Tables 6A-6C. The relative changes reflect the nicotine or total alkaloid levels in quintuple pmt mutants compared to their controls. TIFF2025061515000049.tif83128

[0220] (Table 8A) List of mutations obtained with various genotype combinations of the five PMT genes: single gene mutations TIFF2025061515000050.tif33128

[0221] (Table 8B) List of mutants obtained with various genotype combinations of the five PMT genes: double-gene mutations TIFF2025061515000051.tif59128

[0222] Table 8C: List of mutants obtained with various genotype combinations of the five PMT genes: triple gene combinations TIFF2025061515000052.tif59128

[0223] Table 8D: List of mutants obtained with various genotype combinations of the five PMT genes: quadruple gene combinations TIFF2025061515000053.tif33128

[0224] Table 8E: List of mutants obtained with various genotype combinations of the five PMT genes: quintuple gene combinations TIFF2025061515000054.tif12128

[0225] Example 7: PMT genome editing and tobacco strain development Additional PMT knockout mutants were generated by editing all five PMT genes (PMT1a, PMT1b, PMT2, PMT3, and PMT4) in different tobacco strains. Tobacco protoplasts were transfected using polyethylene glycol (PEG) with a plasmid encoding the genome editing technology (GET2) protein and a specific guide RNA (gRNA) targeting the PMT gene at the desired location. Table 9 lists the gRNA sequences used for PMT editing. Some gRNAs (e.g., numbers 6 and 7) were pooled together to target multiple PMT genes in a single transfection.

[0226] (Table 9) Guide RNAs for GET2 used in Example 7. "Y" indicates that the gRNA can target that PMT gene, while "-" indicates that the gRNA does not target that PMT gene. TIFF2025061515000055.tif49161

[0227] The transfected protoplasts are then immobilized on 1% agarose beads and subjected to tissue culture. Once the calli have grown to approximately 1 mm in diameter, they are plated onto TOM2 plates. The calli are screened for insertions or deletions (indels) at the target location using fragment analysis. Candidates showing a size shift compared to the wild-type control are selected for further culture, and the resulting shoots are again tested by fragment analysis to confirm the presence of indels. Rooted shoots are potted and sequenced at the target location to determine the exact deleted sequence. Young leaves from each plant are harvested and PCR-amplified for PMT fragments using the phirekit. The PMT library for each line is indexed, and 384 lines are pooled and sequenced using Miseq.

[0228] SNP analysis was performed to determine both the sequence of the precise edited pmt mutant alleles and the zygosity status at each PMT locus. Table 10 provides sequence information for indels in each edited line of various tobacco varieties (e.g., Basma, K326, Katerini, TN90, Izmir).

[0229] Table 10: Mutant pmt alleles of various strains generated by genome editing using GET2. The location of each edited site (e.g., indel) is relative to the 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, and SEQ ID NO: 10 for PMT4). For sequences longer than 10 nucleotides, a SEQ ID NO is assigned and shown. TIFF2025061515000056.tif181128TIFF2025061515000057.tif181130TIFF2025061515000058.tif181128

[0230] Table 11 provides the length (in nucleotides) of each PMT indel for each gene in each strain as provided in Table 10.

[0231] Table 11: Length (in nucleotides) of each indel in selected strains provided in Table 10 TIFF2025061515000059.tif186146

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

[0233] (Table 12A) List of exemplary mutant alleles obtained in the PMT1a gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT1a gene with the editing site in the center 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. The lowercase letters in the reference allele sequence (SEQ ID NO: 6) indicate the nucleotides deleted in the mutant allele. TIFF2025061515000060.tif25588TIFF2025061515000061.tif255115TIFF20250615150 00062.tif255119TIFF2025061515000063.tif255119TIFF2025061515000064.tif255100

[0234] (Table 12B) List of exemplary mutant alleles obtained in the PMT1b gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT1b gene with the editing site in the center 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. The lowercase letters in the reference allele sequence (SEQ ID NO: 7) indicate the nucleotides deleted in the mutant allele. TIFF2025061515000065.tif255103TIFF2025061515000066.tif255119TIFF2025061515 000067.tif255119TIFF2025061515000068.tif255119TIFF2025061515000069.tif25588

[0235] (Table 12C) List of exemplary mutant alleles obtained in the PMT2 gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT2 gene with the editing site in the center 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. The lowercase letters in the reference allele sequence (SEQ ID NO: 8) indicate the nucleotides deleted in the mutant allele. TIFF2025061515000070.tif255104TIFF2025061515000071.tif255119TIFF2025061515 000072.tif255119TIFF2025061515000073.tif255119TIFF2025061515000074.tif25588

[0236] (Table 12D) List of exemplary mutant alleles obtained in the PMT3 gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT3 gene with the editing site in the center 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. The lowercase letters in the reference allele sequence (SEQ ID NO: 9) indicate the nucleotides deleted in the mutant allele. TIFF2025061515000075.tif255104TIFF2025061515000076.tif255119TIFF2025061515 000077.tif255119TIFF2025061515000078.tif255119TIFF2025061515000079.tif25588

[0237] (Table 12E) List of exemplary mutant alleles obtained in the PMT4 gene. The sequences of the mutant alleles listed herein represent approximately 90 nucleotides of genomic sequence from each edited PMT4 gene with the editing site in the center 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. The lowercase letters in the sequence of the reference allele (SEQ ID NO: 10) indicate the nucleotides deleted in the mutant allele. TIFF2025061515000080.tif255104TIFF2025061515000081.tif255119TIFF2025061515 000082.tif255119TIFF2025061515000083.tif255119TIFF2025061515000084.tif25588

[0238] Example 8. Alkaloid analysis of PMT-edited strains The homozygous genome-edited tobacco lines from Example 7, as well as control lines, are grown in the field. At the flowering stage, the plants are deflowered, and two weeks after deflowering, leaf blade samples from the third, fourth, and fifth leaves from the top of the plants are collected and alkaloid levels are measured (see Tables 13A-13C). The measurements are performed according to CORESTA Method No. 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatography Analysis (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 (amended 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 percent (wt%) either as is or on a dry weight basis. To report data on a dry weight basis, oven volatiles (OV) determination is required. Unless otherwise specified, total or individual alkaloid or nicotine levels presented herein are on a dry weight basis (e.g., percent total alkaloids or percent nicotine).

[0240] The plants will also be planted in the field, harvested, and tested for alkaloid and TSNA levels in the cured tobacco. Both leaf yield and leaf grade will also be evaluated for the PMT-edited plants.

[0241] Table 13A. Nicotine analysis of K326 and TN90 PMT-edited lines at 2 weeks post-flowering TIFF2025061515000085.tif20873

[0242] Table 13B. Nicotine analysis of K326 and TN90 PMT-edited lines 2 weeks after flower thinning TIFF2025061515000086.tif21270

[0243] Table 13C. Nicotine analysis of Katerini and Basma PMT-edited lines at 2 weeks after flowering TIFF2025061515000087.tif118128

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

[0245] Table 14. PMT-edited ultra-low nicotine male sterile lines TIFF2025061515000088.tif91128

[0246] Example 10. PMT-edited strains are resistant to mold during drying Tobacco leaves harvested from several low-alkaloid tobacco strains are subjected to standard air-curing procedures. After curing is complete, the tobacco leaves are inspected for mold.

[0247] Tobacco derived from LA BU21 exhibits more mold infection than TN90 LC, a TN90 cultivar containing an RNAi construct downregulating all five PMT genes, a TN90 cultivar containing an RNAi construct downregulating the alkaloid biosynthetic gene PR50, and four PMT-edited lines (CS47, CS59, CS63, and CS64) within the TN90 genetic background (see Table 15 and Figures 6A-6E and 7).

[0248] Table 15: Mold damage by tobacco strain. "G" indicates little / no mold, "S" indicates some mold, and "B" indicates significant mold. Mold percentage refers to the proportion of air-dried sticks of tobacco that exhibit each category of mold damage. TIFF2025061515000089.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 <160> 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 tctcttttt 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 ggtgtccaaa 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 tttcagagt ttagcgcatt atggccaggt 1020 tagtactgag aagaaactc aaatgcatat ttaagttaa attgttagg ctatatag 1080 gagttgaat 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 agtagaaag 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 &l...

Claims

1. 1. A tobacco plant or 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, is capable of producing leaves comprising a nicotine level that is lower than the nicotine level of leaves from a control tobacco plant not having the one or more mutant alleles.

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

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

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

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

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

7. 7. The tobacco plant or part thereof of any one of claims 1 to 6, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less 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 in leaves from a control tobacco plant when grown and processed under equivalent conditions.

8. 8. The tobacco plant or part thereof of claim 7, wherein the tobacco plant is capable of producing leaves containing a total alkaloid level that is less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid level of leaves from the control tobacco plant when grown and processed under equivalent conditions.

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

10. 10. The tobacco plant or part thereof of claim 1, wherein the one or more mutant alleles comprise 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 are selected from the group consisting of: PMT protein truncation, non-translated PMT gene transcripts, non-functional PMT proteins, premature 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 produces one or more of the following:

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

13. 13. The tobacco plant or part thereof of any one of claims 1 to 12, wherein the one or more mutant alleles comprise a zygosity selected from the group consisting of homozygous, heterozygous, and heteroallelic.

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

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

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

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

18. 10. The tobacco plant or part thereof of any one of the preceding claims, wherein the tobacco plant is capable of producing 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. 10. The tobacco plant or part thereof of any one of the preceding claims, wherein the tobacco plant is capable of producing 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. 10. The tobacco plant or part thereof of any one of the preceding claims, wherein the tobacco plant is capable of producing cured leaves comprising 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.

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

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

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

24. Contains tobacco leaves, the tobacco leaves exhibit reduced mold infection compared to a control cured tobacco material from cultivar LA Burley 21; 23. The dried tobacco material of claim 22.

25. 23. A tobacco blend comprising the dry tobacco material of claim 22.

26. 26. The tobacco blend of claim 25, wherein the dry 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 dry tobacco in the tobacco blend.

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

28. A tobacco product comprising the dried tobacco material of claim 22.

29. 29. The tobacco product of claim 28, wherein the tobacco product is 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. 30. The tobacco product of claim 28, which is a smokeless tobacco product.

31. 31. The tobacco product of 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. 23. A reconstituted tobacco comprising the dried tobacco material of claim 22.

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