COMPOSITIONS AND METHODS BASED ON PMT ENGINEERING FOR PRODUCING TOBACCO PLANTS AND PRODUCTS HAVING ALTERED ALKALOID LEVELS

Description

CROSS-REFERENCE TO REPLATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTING

This application claims the benefit of U.S. Provisional Application No. 62/703,775, filed Jul. 26, 2019 and U.S. Provisional Application No. 62/848,159, filed May 15, 2019, which are incorporated by reference in its entirety herein. A sequence listing contained in the file named “P34620US02_SL.txt” which is 200,338 bytes (measured in MS-Windows®) and created on Jul. 26, 2019, is filed electronically herewith and incorporated by reference in its entirety.

FIELD

The present disclosure provides tobacco genetic engineering for modulating alkaloid and nicotine levels.

BACKGROUND

Nicotine is the predominant alkaloid, usually accounting for more than 90-95% of the total alkaloids in commercial tobacco cultivars. The remaining alkaloid fraction is primarily comprised three additional alkaloids: nornicotine, anabasine, and anatabine. Tobacco plants with reduced nicotine levels have been achieved with varying and inconsistent results by modulating different nicotine biosynthetic genes and transcriptional regulators. There is a need for new technologies to reduce nicotine levels in tobacco leaves.

SUMMARY

The present disclosure provides tobacco plants with altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants.

In an aspect, 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, wherein the tobacco plant is capable of producing a leaf comprising a nicotine level less than the nicotine level of a leaf from a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions.

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

In an 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 to 8E.

In an aspect, the present disclosure provides a tobacco plant as listed in Tables 4A to 4E or Table 10. In another aspect, the present disclosure provides a progeny plant of a tobacco plant in Tables 4A to 4E or Table 10, from either selfing or a cross with another plant in Tables 4A to 4E or Table 10.

In another aspect, the present disclosure provides a tobacco plant comprising various combinations of the pmt mutant alleles listed in Tables 5A to 5E or Tables 12A to 12E to give rise to a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. In an aspect, the present disclosure provides a tobacco plant comprising a pmt mutant allele sequence selected from the group consisting of SEQ ID Nos. 21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697.

The present disclosure further provides cured tobacco, tobacco blends, tobacco products comprising plant material from tobacco plants, lines, varieties or hybrids disclosed.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID Nos: 1 to 5 set forth exemplary genomic sequences of PMT1b, PMT1a, PMT2, PMT3 and PMT4, respectfully, from a TN90 reference genome.

SEQ ID Nos: 6 to 10 set forth exemplary cDNA sequences of PMT1b, PMT1a, PMT2, PMT3 and PMT4, respectfully, from TN90.

SEQ ID Nos: 11 to 15 set forth exemplary polypeptide sequences of PMT1b, PMT1a, PMT2, PMT3 and PMT4, respectfully, from TN90.

SEQ ID Nos: 16 to 22 set forth exemplary guide RNA sequences.

SEQ ID Nos: 23 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697 set forth exemplary edited pmt mutant sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RNA expression of five PMT genes in TN90 roots

FIG. 2: Nicotine levels in various low-alkaloid lines: CS15 (a quintuple pmt knock-out mutant line CS15 in the NLM (Ph Ph) background), a PMT RNAi transgenic line in the VA359 background) and a low-nicotine KY171 (“LN KY171”) variety (the KY 171 background harboring nic1 and nic2 double mutations), in comparison to their respective normal-alkaloid control line: NLM (Ph Ph), VA359, and KY171 background.

FIG. 3: Total alkaloid levels in various low-alkaloid lines: CS15, PMT RNAi, and LN KY171, in comparison to their respective normal-alkaloid control line: NLM (Ph Ph), VA359, and KY171 background.

FIG. 4: Leaf yield in various low-alkaloid lines: CS15, PMT RNAi, and LN KY171, in comparison to their respective normal-alkaloid control line: NLM (Ph Ph), VA359, and KY171 background.

FIG. 5: Leaf quality in various low-alkaloid lines: CS15, PMT RNAi, and LN KY171, in comparison to their respective normal-alkaloid control line: NLM (Ph Ph), VA359, and KY171 background.

Photographs depicting mold growth on cured tobacco, including TN90 LC (FIG. 6A), LA BU 21 (FIG. 6B), TN90 comprising an RNAi construct to downregulate PR50 (FIG. 6C), TN90 comprising an RNAi construct to downregulate PMT genes (FIG. 6D), and TN90 comprising edits to all five PMT genes (FIG. 6E).

FIG. 7: Depiction of mold infection observed in the lines examined in FIGS. 6A-6E.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. One skilled in the art will recognize many methods can be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described. For purposes of the present disclosure, the following terms are defined below.

Any references cited herein, including, e.g., all patents and publications are incorporated by reference in their entirety.

As used herein, the singular form “a,” “an,’ and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth by 10%.

As used herein, phrases such as “less than”, “more than”, “at least”, “at most”, “approximately”, “below”, “above”, and “about”, when used in conjunction with a series of numerical values, modify each and every value within the series. For example, an expression of “less than 1%, 2%, or 3%” is equivalent to “less than 1%, less than 2%, or less than 3%.”

As used herein, a tobacco plant refers to a plant from the species Nicotiana tabacum.

Nicotine biosynthesis in tobacco starts with the methylation of the polyamine, putrescine, to N-methylputrescine by the enzyme, putrescine N-methyltransferase (PMT), using S-adenosyl-methionine as the co-factor. This is a step that commits precursor metabolites to nicotine biosynthesis. PMT enzymes are classified under the enzyme classification system as EC 2.1.1.53. In Nicotiana tabacum, five genes encode putrescine N-methyltransferases, designated PMT1a, PMT1b, PMT2, PMT3, and PMT4. Table 1A lists genomic DNA sequences, cDNA sequences, and protein sequences of these five PMT genes in a TN90 plant. The present disclosure describes compositions and methods that are used to edit PMT genes to produce pmt mutant plants having reduced nicotine levels while maintaining leaf quality.

As used herein, “PMT1b” or the “PMT1b gene” refers to a genic locus in tobacco encoding a polypeptide having an exemplary amino acid sequence in TN90 as set forth in SEQ ID No. 11.

As used herein, “PMT1a” or the “PMT1a gene” refers to a genic locus in tobacco encoding a polypeptide having an exemplary amino acid sequence in TN90 as set forth in SEQ ID No. 12.

As used herein, “PMT2” or the “PMT2 gene” refers to a genic locus in tobacco encoding a polypeptide having an exemplary amino acid sequence in TN90 as set forth in SEQ ID No. 13.

As used herein, “PMT3” or the “PMT3 gene” refers to a genic locus in tobacco encoding a polypeptide having an exemplary amino acid sequence in TN90 as set forth in SEQ ID No. 14.

As used herein, “PMT4” or the “PMT4 gene” refers to a genic locus in tobacco encoding a polypeptide having an exemplary amino acid sequence in TN90 as set forth in SEQ ID No. 15.

As used herein, a mutation refers to an inheritable genetic modification introduced into a gene to reduce, inhibit, or eliminate the expression or activity of a product encoded by the gene. Such a modification can be in any sequence region of a gene, for example, in a promoter, 5′ UTR, exon, intron, 3′ UTR, or terminator region. In an aspect, mutations are not natural polymorphisms that exist in a particular tobacco variety or cultivar. As used herein, a “mutant allele” refers to an allele from a locus where the allele comprises a mutation.

As used herein, a “pmt mutant” refers to a tobacco plant comprising one or more mutations in one or more PMT genes. A pmt mutant can be a single mutant, a double mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. As used herein, a single, double, triple, quadruple, or quintuple pmt mutant refers to a mutant having modifications in one, two, three, four, or five PMT genes, respectively. A pmt mutant can also be a homozygous mutant, a heterozygous mutant, or a heteroallelic mutant combination in one or more PMT genes.

As used herein, a gene name or a genic locus name is capitalized and shown in italic, e.g., PMT1a, PMT1b, PMT2, PMT3, and PMT4. A protein or polypeptide name is capitalized without being italicized, e.g., PMT1a, PMT1b, PMT2, PMT3, and PMT4. A mutant name (for either referencing to a general mutation in a gene or a group of genes, or referencing to a specific mutant allele) is shown in lower case and italic, e.g., pmt, pmt1a, pmt1b, pmt2, pmt3, and pmt4.

In an aspect, 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, wherein the tobacco plant is capable of producing a leaf comprising a nicotine level less than the nicotine level of a leaf from a control tobacco plant not having the one or more mutant alleles when grown and processed under comparable conditions. In an aspect, a single pmt mutant tobacco plant is provided. In another aspect, a single pmt mutant tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below 90%, or below 95% of the nicotine level of a control plant not having the single pmt mutation when grown in similar growth conditions. In a further aspect, a single pmt mutant tobacco plant comprises nicotine at a level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the nicotine level of a control plant not having the single pmt mutation when grown in similar growth conditions.

In another aspect, a 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 an aspect, a double pmt mutant tobacco plant is provided. In another aspect, a double pmt mutant tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below 90%, or below 95% of the nicotine level of a control plant not having the double pmt mutations when grown in similar growth conditions. In a further aspect, a double pmt mutant tobacco plant comprises nicotine at a level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the nicotine level of a control plant not having the double pmt mutations when grown in similar growth conditions.

In a further aspect, a 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 an aspect, a triple pmt mutant tobacco plant is provided. In another aspect, a triple pmt mutant tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below 90%, or below 95% of the nicotine level of a control plant not having the triple pmt mutations when grown in similar growth conditions. In a further aspect, a triple pmt mutant tobacco plant comprises nicotine at a level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the nicotine level of a control plant not having the triple pmt mutations when grown in similar growth conditions.

In another aspect, a 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 an aspect, a quadruple pmt mutant tobacco plant is provided. In another aspect, a quadruple pmt mutant tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below 90%, or below 95% of the nicotine level of a control plant not having the quadruple pmt mutations when grown in similar growth conditions. In a further aspect, a quadruple pmt mutant tobacco plant comprises nicotine at a level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the nicotine level of a control plant not having the quadruple pmt mutations when grown in similar growth conditions.

In a further aspect, a tobacco plant comprises one or more mutant alleles in five PMT genes selected from the group consisting of PMT1a, PMT1b, PMT2, PMT3, and PMT4. In an aspect, a quintuplepmt mutant tobacco plant is provided. In another aspect, a quintuplepmt mutant tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, below 80%, below 90%, or below 95% of the nicotine level of a control plant not having the quintuple pmt mutations when grown in similar growth conditions. In a further aspect, a quintuple pmt mutant tobacco plant comprises nicotine at a level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the nicotine level of a control plant not having the quintuple pmt mutations when grown in similar growth conditions.

In an aspect, a tobacco plant is a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a quintuple mutant as listed in Tables 8A to 8E. In another aspect, a tobacco plant comprises one or more pmt mutant alleles listed in Tables 5A to 5E and Tables 12A to 12E. Each and every combination of the pmt mutant alleles listed in Tables 5A to 5E and Tables 12A to 12E is also provided to give rise to a single pmt mutant, a double pmt mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. Each of the mutated loci can be either homozygous or heterozygous, or comprises a heteroallelic combination. In another aspect, a tobacco plant comprises a pmt mutant genotype combination as shown for each individual line listed in Tables 4A to 4E and Table 10. In an aspect, a tobacco plant comprises a pmt mutant allele sequence selected from the group consisting of SEQ ID Nos. 21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697. In another aspect, the present disclosure provides a double pmt mutant, a triple mutant, a quadruple mutant, or a quintuple mutant comprising pmt mutant allele sequences selected from the group consisting of SEQ ID Nos. 21 to 200, 410 to 441, 474 to 505, 538 to 569, 602 to 633, and 666 to 697.

In an aspect, a tobacco plant is capable of producing a leaf comprising a nicotine level 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 a leaf from a control tobacco plant when grown and processed under comparable conditions. In another aspect, a tobacco plant is capable of producing a leaf comprising a nicotine level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the nicotine level of a control tobacco plant when grown and processed under comparable conditions.

In another aspect, a tobacco plant is capable of producing a leaf comprising a total alkaloid level 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 a leaf from a control tobacco plant when grown and processed under comparable conditions. In another aspect, a tobacco plant is capable of producing a leaf comprising a total alkaloid level between 1% and 5%, between 5% and 10%, between 10% and 20%, between 20% and 30%, between 30% and 40%, between 40% and 50%, between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 95% of the total alkaloid level of a control tobacco plant when grown and processed under comparable conditions.

In a further aspect, a tobacco plant is capable of producing a leaf comprising a total alkaloid level less than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total alkaloid level of a leaf from a control tobacco plant when grown and processed under comparable conditions.

In an aspect, a mutant pmt allele comprises a mutation in a PMT sequence region selected from the group consisting of a promoter, 5′ UTR, first exon, first intron, second exon, second intron, third exon, third intron, fourth exon, fourth intron, fifth exon, fifth intron, sixth exon, sixth intron, seventh exon, seventh intron, eighth exon, 3′ UTR, terminator, and any combination thereof. In another aspect, a mutant pmt allele comprises a mutation in a PMT genomic sequence region listed in Tables 1D to 1H.

In another aspect, a mutant pmt allele comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof. In an aspect, a mutant pmt allele is a null allele or a knock-out allele.

In an aspect, a mutant pmt allele results in one or more of the following: a PMT protein truncation, a non-translatable PMT gene transcript, a non-functional PMT protein, a premature stop codon in a PMT gene, and any combination thereof.

In another aspect, a mutant pmt allele comprises a mutation selected from the group consisting of a substitution, a deletion, an insertion, a duplication, and an inversion of one or more nucleotides relative to a wild-type PMT gene.

In an aspect, a pmt mutant comprises a zygosity status selected from the group consisting of homozygous, heterozygous, and heteroallelic. In another aspect, a pmt mutant is homozygous or heteroallelic in at least 1, 2, 3, 4, or 5 PMT genes. In an aspect, a pmt mutant is homozygous or heteroallelic in at least 4 PMT genes. In another aspect, a pmt mutant is homozygous or heteroallelic in all five PMT genes. In another aspect, a pmt mutant comprises mutations in PMT1a and PMT3.

In an aspect, a tobacco plant is capable of producing a leaf comprising 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% dry weight.

In another aspect, a tobacco plant is capable of producing a leaf comprising 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% dry weight.

In a further aspect, a tobacco plant is capable of producing a cured leaf comprising a total TSNA level of between 2 and 0.05, between 1.9 and 0.05, between 1.8 and 0.05, between 1.7 and 0.05, between 1.6 and 0.05, between 1.5 and 0.05, between 1.4 and 0.05, between 1.3 and 0.05, between 1.2 and 0.05, between 1.1 and 0.05, between 1.0 and 0.05, between 0.9 and 0.05, between 0.8 and 0.05, between 0.7 and 0.05, between 0.6 and 0.05, between 0.5 and 0.05, between 0.4 and 0.05, between 0.3 and 0.05, between 0.2 and 0.05, between 0.15 and 0.05, or between 0.1 and 0.05 ppm.

In an aspect, a tobacco plant is capable of producing leaves, when cured, having a USDA grade index value selected from the group consisting of 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another aspect, a tobacco plant is capable of producing leaves, when cured, having a USDA grade index value comparable to that of a control plant when grown and cured in similar conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except for the modification. In a further aspect, a tobacco plant is capable of producing leaves, when cured, 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 a control plant when grown in similar conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except the modification. In a further aspect, a tobacco plant is capable of producing leaves, when cured, having a USDA grade index value of between 65% and 130%, between 70% and 130%, between 75% and 130%, between 80% and 130%, between 85% and 130%, between 90% and 130%, between 95% and 130%, between 100% and 130%, between 105% and 130%, between 110% and 130%, between 115% and 130%, or between 120% and 130% of the USDA grade index value of a control plant. In a further aspect, a tobacco plant is capable of producing leaves, when cured, having a USDA grade index value of between 70% and 125%, between 75% and 120%, between 80% and 115%, between 85% and 110%, or between 90% and 100% of the USDA grade index value of a control plant.

In an aspect, a tobacco plant comprises nicotine at a level below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the nicotine level of a control plant when grown in similar growth conditions, where the control plant shares an essentially identical genetic background with the tobacco plant except for the modification.

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

In an aspect, a tobacco plant comprises one or more pmt mutant alleles and further comprises a mutation in an ERF gene of Nic2 locus. In an aspect, a 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 an aspect, a tobacco plant further comprises one or more mutations in ERF189, ERF115, or both.

In an aspect, a tobacco plant comprises one or more qpt mutant alleles and further comprises a mutation in an ERF gene of Nic1 locus (or Nic1b locus as in PCT/US2019/013345 filed on Jan. 11, 2019, published as WO/2019/140297). See also WO/2018/237107. In an aspect, a 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 an aspect, a 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.

In an aspect, the present disclosure further provides apmt mutant tobacco plant, or part thereof, comprising a nicotine 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%, where the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value of 50 or more 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and 95 or more. In another aspect, such pmt mutant tobacco plant comprises a nicotine level of less than 0.02% and are capable of producing leaves, when cured, having a USDA grade index value of 70 or more. In a further aspect, such tobacco plant comprises a nicotine level of less than 0.01% and are capable of producing leaves, when cured, having a USDA grade index value of 70 or more.

In an aspect, the present disclosure also provides a tobacco plant, or part thereof, comprising a non-transgenic mutation, where the non-transgenic mutation reduces the nicotine or total alkaloid level of the tobacco plant to below 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80% of the nicotine level of a control plant when grown in similar growth conditions, where the tobacco plant is capable of producing leaves, when cured, having a USDA grade index value comparable to the USDA grade index value of the control plant, and where the control plant shares an essentially identical genetic background with the tobacco plant except the non-transgenic mutation.

In an aspect, a tobacco plant comprises a pmt mutation introduced by an approach selected from the group consisting of random mutagenesis and targeted mutagenesis. In another aspect, a pmt mutation is introduced by a targeted mutagenesis approach selected from the group consisting of meganuclease, zinc finger nuclease, TALEN, and CRISPR.

Unless specified otherwise, measurements of alkaloid or nicotine levels (or another leaf chemistry or property characterization) or leaf grade index values mentioned herein for a tobacco plant, variety, cultivar, or line refer to average measurements, including, for example, an average of multiple leaves of a single plant or an average measurement from a population of tobacco plants from a single variety, cultivar, or line.

Unless specified otherwise, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pooled leaf sample collected from leaf number 3, 4, and 5 after topping. As used herein, whenever a comparison between leaves from two plants (e.g., a mutant plant versus a control plant) is mentioned, leaves from the same or comparable stalk position(s) and developmental stage(s) are intended so that the comparison can demonstrate effects due to genotype differences, not from other factors. As an illustration, leaf 3 of a wild-type control plant is intended as a reference point for comparing with leaf 3 of a pmt mutant plant. In an aspect, a tobacco plant comprising at least one pmt mutation is compared to a control tobacco plant of the same tobacco variety.

Nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant can also be measured in alternative ways. In an aspect, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a leaf having the highest level of nicotine or alkaloid (or another leaf chemistry or property characterization). In an aspect, the nicotine or alkaloid level of a tobacco plant is measured after topping in leaf number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In another aspect, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pool of two or more leaves with consecutive leaf numbers selected from the group consisting of leaf number 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 aspect, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a leaf with a leaf number selected from the group consisting of between 1 and 5, between 6 and 10, between 11 and 15, between 16 and 20, between 21 and 25, and between 26 and 30. In another aspect, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pool of two or more leaves with leaf numbers selected from the group consisting of between 1 and 5, between 6 and 10, between 11 and 15, between 16 and 20, between 21 and 25, and between 26 and 30. In another aspect, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pool of three or more leaves with leaf numbers selected from the group consisting of between 1 and 5, between 6 and 10, between 11 and 15, between 16 and 20, between 21 and 25, and between 26 and 30.

As used herein, leaf numbering is based on the leaf position on a tobacco stalk with leaf number 1 being the youngest leaf (at the top) after topping and the highest leaf number assigned to the oldest leaf (at the bottom).

A population of tobacco plants or a collection of tobacco leaves for determining an average measurement (e.g., alkaloid or nicotine level or leaf grading) can be of any size, for example, 5, 10, 15, 20, 25, 30, 35, 40, or 50. Industry-accepted standard protocols are followed for determining average measurements or grad index values.

As used herein, “topping” refers to the removal of the stalk apex, including the SAM, flowers, and up to several adjacent leaves, when a tobacco plant is near vegetative maturity and around the start of reproductive growth. Typically, tobacco plants are topped in the button stage (soon after the flower begins to appear). For example, greenhouse or field-grown tobacco plants can be topped when 50% of the plants have at least one open flower. Topping a tobacco plant results in the loss of apical dominance and also induces increased alkaloid production.

Unless indicated otherwise, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured 2 weeks after topping. Alternatively, other time points can be used. In an aspect, the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured about 1, 2, 3, 4, or 5 weeks after topping. In another aspect, the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization) of a tobacco plant is measured about 3, 5, 7, 10, 12, 14, 17, 19 or 21 days after topping.

As used herein, “similar growth conditions” or “comparable growth conditions” refer to similar environmental conditions and/or agronomic practices for growing and making meaningful comparisons between two or more plant genotypes so that neither environmental conditions nor agronomic practices would contribute to or explain any difference observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrition (e.g., nitrogen and phosphorus). Agronomic practices include, for example, seeding, clipping, undercutting, transplanting, topping, and suckering. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103.

“Alkaloids” are complex, nitrogen-containing compounds that naturally occur in plants, and have pharmacological effects in humans and animals. “Nicotine” is the primary natural alkaloid in commercialized cigarette tobacco and accounts for about 90 percent 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-methyl anatabine, N-methyl anabasine, pseudooxynicotine, 2,3 dipyridyl and others.

Alkaloid levels can be assayed by methods known in the art, for example by quantification based on gas-liquid chromatography, high performance liquid chromatography, radio-immunoassays, and enzyme-linked immunosorbent assays. For example, nicotinic alkaloid levels can be measured by a GC-FID method based on CORESTA Recommended Method No. 7, 1987 and ISO Standards (ISO TC 126N 394 E. See also Hibi et al., Plant Physiology 100: 826-35 (1992) for a method using gas-liquid chromatography equipped with a capillary column and an FID detector.

Unless specifically indicated otherwise, alkaloids and nicotine levels are measured using a method in accordance with CORESTA Method No 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and those defined in the Centers for Disease Control and Prevention's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, as published in the Federal Register Vol. 64, No. 55 Mar. 23, 1999 (and as amended in Vol. 74, No. 4, Jan. 7, 2009). Alternatively, tobacco total alkaloids can be measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, Pa.) and described by Collins et al., Tobacco Science 13:79-81 (1969). In short, samples of tobacco can be dried, ground, and extracted prior to analysis of total alkaloids and reducing sugars. The method then employs an acetic acid/methanol/water extraction and charcoal for decolorization. Determination of total alkaloids is based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of an aromatic amine to form a colored complex which is measured at 460 nm. Unless specified otherwise, total alkaloid levels or nicotine levels shown herein are on a dry weight basis (e.g., percent total alkaloid or percent nicotine).

In an aspect, a tobacco plant comprises an average nicotine or total alkaloid level selected from the group consisting of 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% on a dry weight basis. In another aspect, a tobacco plant comprises an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.02%, between 0.02% and 0.05%, between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%, between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%, between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%, between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%, between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between 1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between 1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between 1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1% and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and 2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and 2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%, between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%, between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis. In a further aspect, a tobacco plant comprises an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.1%, between 0.02% and 0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%, between 0.05% and 0.5%, between 0.75% and 1%, between 0.1% and 1.5%, between 0.15% and 2%, between 0.2% and 3%, and between 0.3% and 3.5% on a dry weight basis.

The present disclosure also provides a tobacco plant having an altered nicotine level without negative impacts over other tobacco traits, e.g., leaf grade index value. In an aspect, a low-nicotine or nicotine-free tobacco variety provides cured tobacco of commercially acceptable grade.

Tobacco grades are evaluated based on factors including, but not limited to, the leaf stalk position, leaf size, leaf color, leaf uniformity and integrity, ripeness, texture, elasticity, sheen (related with the intensity and the depth of coloration of the leaf as well as the shine), hygroscopicity (the faculty of the tobacco leaves to absorb and to retain the ambient moisture), and green nuance or cast. Leaf grade can be determined, for example, using an Official Standard Grade published by the Agricultural Marketing Service of the US Department of Agriculture (7 U.S.C. § 511). See, e.g., Official Standard Grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93), effective Nov. 5, 1990 (55 F.R. 40645); Official Standard Grades for Flue-Cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective Mar. 27, 1989 (54 F.R. 7925); Official Standard Grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective Jan. 8, 1965 (29 F.R. 16854); Official Standard Grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44), effective Dec. 8, 1963 (28 F.R. 11719 and 28 F.R. 11926); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55), effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), Effective April 1971. A USDA grade index value can be determined according to an industry accepted grade index. See, e.g., Bowman et al, Tobacco Science, 32:39-40(1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, Jul. 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern., 192:55-57 (all foregoing references are incorporated by inference in their entirety). In an aspect, a USDA grade index is a 0-100 numerical representation of federal grade received and is a weighted average of all stalk positions. A higher grade index indicates higher quality. Alternatively, leaf grade can be determined via hyper-spectral imaging. See e.g., WO 2011/027315 (published on Mar. 10, 2011, and incorporated by inference in its entirety).

In an aspect, a tobacco plant provided herein comprises a similar level of one or more tobacco aroma compounds compared to a control tobacco plant when grown in similar growth conditions. In another aspect, a tobacco plant provided herein comprise a similar level of one or more tobacco aroma compounds selected from the group consisting of 3-methylvaleric acid, valeric acid, isovaleric acid, a labdenoid, a cembrenoid, a sugar ester, and a reducing sugar, compared to a control tobacco plant when grown in similar growth conditions.

As used herein, tobacco aroma compounds are compounds associated with the flavor and aroma of tobacco smoke. These compounds include, but are not limited to, 3-methylvaleric acid, valeric acid, isovaleric acid, cembrenoid and labdenoid diterpenes, and sugar esters. Concentrations of tobacco aroma compounds can be measured by any known metabolite profiling methods in the art including, without limitation, gas chromatography mass spectrometry (GC-MS), Nuclear Magnetic Resonance Spectroscopy, liquid chromatography-linked mass spectrometry. See The Handbook of Plant Metabolomics, edited by Weckwerth and Kahl, (Wiley-Blackwell) (May 28, 2013).

As used herein, “reducing sugar(s)” are any sugar (monosaccharide or polysaccharide) that has a free or potentially free aldehdye or ketone group. Glucose and fructose act as nicotine buffers in cigarette smoke by reducing smoke pH and effectively reducing the amount of “free” unprotonated nicotine. Reducing sugars balances smoke flavor, 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 across tobacco varieties, within the same variety, and within the same plant line caused by planting conditions. Reducing sugar levels can be measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co (West Chester, Pa.) and described by Davis, Tobacco Science 20:139-144 (1976). For example, a sample is dialyzed against a sodium carbonate solution. Copper neocuproin is added to the sample and the solution is heated. The copper neocuproin chelate is reduced in the presence of sugars resulting in a colored complex which is measured at 460 nm.

In an aspect, a tobacco plant comprises one or more non-naturally existing mutant alleles in one or more PMT gene loci which reduce or eliminate PMT enzymatic activity from the one or more PMT gene loci. In an aspect, these mutant alleles result in lower nicotine levels. Mutant pmt alleles can be introduced by any method known in the art including random or targeted mutagenesis approaches.

Such mutagenesis methods include, without limitation, treatment of seeds 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, for example, Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; and Poehlman, Breeding Field Crops, Van Nostrand Reinhold, New York (3.sup.rd ed), 1987), transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and 5,013,658), as well as T-DNA insertion methodologies (Hoekema et al., 1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists of chemically inducing random point mutations over the length of the genome. Fast neutron mutagenesis consists of exposing seeds to neutron bombardment which causes large deletions through double stranded DNA breakage. Transposon tagging comprises inserting a transposon within an endogenous gene to reduce or eliminate expression of the gene. The types of mutations that may be present in a tobacco gene include, for example, point mutations, deletions, insertions, duplications, and inversions. Such mutations desirably are present in the coding region of a tobacco gene; however mutations in the promoter region, and intron, or an untranslated region of a tobacco gene may also be desirable.

In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), 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 impact gene expression or that interfere with the function of genes can be determined using methods that are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues can be particularly effective in inhibiting the function of a protein. In an aspect, tobacco plants comprise a nonsense (e.g., stop codon) mutation in one or more PMT genes described herein.

It will be appreciated that, when identifying a mutation, the endogenous reference DNA sequence should be from the same variety of tobacco. For example, if a modified tobacco plant comprising a mutation is from the variety TN90, then the endogenous reference sequence must be the endogenous TN90 sequence, not a homologous sequence from a different tobacco variety (e.g., K326). Similarly, if a modified tobacco cell comprising a mutation is a TN90 cell, then 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).

In an aspect, the present disclosure also provides a tobacco line with altered nicotine levels while maintaining commercially acceptable leaf quality. This line can be produced by introducing mutations into one or more PMT genes via precise genome engineering technologies, for example, Transcription activator-like effector nucleases (TALENs), meganuclease, zinc finger nuclease, and a clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/Csm1 system, and a combination thereof (see, for example, U.S. Patent Application publication 2017/0233756). See, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013).

The screening and selection of mutagenized tobacco plants can be through any methodologies known to those having ordinary skill in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide, Northern blots, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, Next Generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454), enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known.

In an aspect, a 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.

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 genome nucleic acid sequence, or removal or replacement of an endogenous plant genome nucleic acid sequence. In an aspect, an 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 with an endogenous nucleic acid sequence. In an aspect, an 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 with a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 10, and fragments thereof. In another aspect, an 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 with a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOs: 11 to 15.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/Csm1 and CRISPR/Cpf1 induce a double-strand DNA break at a target site of a genomic sequence that is then repaired by the natural processes of homologous recombination (HR) or non-homologous end-joining (NHEJ). Sequence modifications then occur at the cleaved sites, which can include deletions or insertions that result in gene disruption in the case of NHEJ, or integration of donor nucleic acid sequences by HR. In an aspect, a method provided comprises editing a plant genome with a nuclease provided to mutate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more than 10 nucleotides in the plant genome via HR with a donor polynucleotide. In an aspect, a mutation provided is caused by genome editing using a nuclease. In another aspect, a mutation provided is caused by non-homologous end-joining or homologous recombination.

Meganucleases, which are commonly identified in microbes, are unique enzymes with high activity and long recognition sequences (>14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). The engineering of meganucleases can be more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.

ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be designed to cleave almost any long stretch of double-stranded DNA for modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain of FokI endonuclease fused to a zinc finger array engineered to bind a target DNA sequence.

The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-finger arrays. The amino acids at positions −1, +2, +3, and +6 relative to the start of the zinc finger ∞-helix, which contribute to site-specific binding to the target DNA, can be changed and customized to fit specific target sequences. The other amino acids form the consensus backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.

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

Without being limited by any scientific theory, because the DNA-binding specificities of zinc finger domains can in principle be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any gene sequence. Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a FokI nuclease domain. When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double-stranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. The Xanthomonas pathogens secrete TALEs into the host plant cell during infection. The TALE moves to the nucleus, where it recognizes and binds to a specific DNA sequence in the promoter region of a specific DNA sequence in the promoter region of a specific gene in the host genome. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of 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 diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity.

A relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill 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.

A CRISPR/Cas9 system, CRISPR/Csm1, or a CRISPR/Cpf1 system are alternatives to the FokI-based methods ZFN and TALEN. The CRISPR systems are based on RNA-guided engineered nucleases that use complementary base pairing to recognize DNA sequences at target sites.

CRISPR/Cas9, CRISPR/Csm1, and a CRISPR/Cpf1 systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. The immunity is acquired by the integration of short fragments of the invading DNA known as spacers between two adjacent repeats at the proximal end of a CRISPR locus. The CRISPR arrays, including the spacers, are transcribed during subsequent encounters with invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs) approximately 40 nt in length, which combine with the trans-activating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease. This cleaves homologous double-stranded DNA sequences known as protospacers in the invading DNA. A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5-NGG-3 but less frequently NAG. Specificity is provided by the so-called “seed sequence” approximately 12 bases upstream of the PAM, which must match between the RNA and target DNA. Cpf1 and Csm1 act in a similar manner to Cas9, but Cpf1 and Csm1 do not require a tracrRNA.

In still another aspect, a tobacco plant provided here comprises one or more pmt mutations and further comprises one or more mutations in one or more loci encoding a nicotine demethylase (e.g., CYP82E4, CYP82E5, CYP82E10) that confer reduced amounts of nornicotine (See U.S. Pat. Nos. 8,319,011; 8,124,851; 9,187,759; 9,228,194; 9,228,195; 9,247,706) compared to a control plant lacking one or more mutations in one or more loci encoding a nicotine demethylase. In an aspect, a tobacco plant described further comprises reduced nicotine demethylase activity compared to a control plant when grown and cured under comparable conditions.

In an aspect, a pmt mutant tobacco plant further comprises a mutation capable of producing a leaf comprising an anabasine level less than the anabasine level of a leaf from a wild-type control tobacco plant when grown and processed under comparable conditions. In another aspect, a pmt mutant tobacco plant further comprises a mutation capable of producing a leaf comprising an anabasine level less than 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the anabasine level of a leaf from a wild-type control tobacco plant when grown and processed under comparable conditions.

In an aspect, a pmt mutant tobacco plant comprises a further mutation capable of producing a leaf comprising a more than 2 fold reduction of the anatabine level compared to a leaf from a control tobacco plant when grown and processed under comparable conditions. In another aspect, a pmt mutant tobacco plant comprises a further mutation capable of producing a leaf comprising a more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 fold reduction of the anatabine level compared to a leaf from a wild-type control tobacco plant when grown and processed under comparable conditions. In an aspect, a mutation providing lower level of anatabine is a mutation described in US Application Publication No. 2014/0283165 and US Application Publication No. 2016/0010103. In another aspect, a pmt mutant further comprises a mutation in a quinolate phosphoribosyl transferase (QPT) or quinolinate synthase (QS) gene. In a further aspect, a pmt mutant plant further comprises a transgene or mutation suppressing the expression or activity of a QPT or QS gene.

In an aspect, a pmt mutant tobacco plant further comprises a mutation capable of providing a nornicotine level less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or 35% of the nornicotine level of a leaf from a wild-type control tobacco plant when grown and processed under comparable conditions.

In an aspect, a pmt mutant tobacco plant is capable of producing a cured leaf comprising a total N-nitrosonornicotine (NNN) level 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.

In another aspect, a pmt mutant tobacco plant is capable of producing a cured leaf comprising a total NNN level of between 2 and 0.05, between 1.9 and 0.05, between 1.8 and 0.05, between 1.7 and 0.05, between 1.6 and 0.05, between 1.5 and 0.05, between 1.4 and 0.05, between 1.3 and 0.05, between 1.2 and 0.05, between 1.1 and 0.05, between 1.0 and 0.05, between 0.9 and 0.05, between 0.8 and 0.05, between 0.7 and 0.05, between 0.6 and 0.05, between 0.5 and 0.05, between 0.4 and 0.05, between 0.3 and 0.05, between 0.2 and 0.05, between 0.15 and 0.05, or between 0.1 and 0.05 parts per million (ppm).

In an aspect, a pmt mutant tobacco plant is capable of producing a cured leaf comprising a total nicotine-derived nitrosamine ketone (NNK) level 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.

In another aspect, a pmt mutant tobacco plant is capable of producing a cured leaf comprising a total NNK level of between 2 and 0.05, between 1.9 and 0.05, between 1.8 and 0.05, between 1.7 and 0.05, between 1.6 and 0.05, between 1.5 and 0.05, between 1.4 and 0.05, between 1.3 and 0.05, between 1.2 and 0.05, between 1.1 and 0.05, between 1.0 and 0.05, between 0.9 and 0.05, between 0.8 and 0.05, between 0.7 and 0.05, between 0.6 and 0.05, between 0.5 and 0.05, between 0.4 and 0.05, between 0.3 and 0.05, between 0.2 and 0.05, between 0.15 and 0.05, or between 0.1 and 0.05 ppm.

In an aspect, a pmt mutant tobacco plant further comprises a mutation or transgene providing an increased level of one or more antioxidants. In another aspect, a pmt mutant tobacco plant further comprises a genetic modification in an endogenous gene and further comprises an increased level of one or more antioxidants in a cured leaf compared to a control cured tobacco leaf lacking the genetic modification, where the endogenous gene encodes an antioxidant biosynthetic enzyme, a regulatory transcription factor of an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In a father aspect, a pmt mutant tobacco plant further comprises a transgene and further comprises an increased level of one or more antioxidants in a cured leaf compared to a control cured tobacco leaf lacking the transgene, where the transgene encodes or directly modulates an antioxidant biosynthetic enzyme, a regulatory transcription factor of an antioxidant, an antioxidant transporter, an antioxidant metabolic enzyme, or a combination thereof. In an aspect, a pmt mutant tobacco plant further comprises a transgene or a 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 aspect, a pmt mutant tobacco plant further comprises one or more transgenes or genetic modification for increasing antioxidants or decreasing one or more TSNAs as described in WIPO Publication No. 2018/067985 or US Publication No. 2018/0119163.

In an aspect, a tobacco plant described is a modified tobacco plant. As used herein, “modified”, in the context of a plant, refers to a plant comprising a genetic alteration introduced for certain purposes and beyond natural polymorphisms.

In an aspect, a tobacco plant described is a cisgenic plant. As used herein, “cisgenesis” or “cisgenic” refers to genetic modification of a plant, plant cell, or plant genome in which all components (e.g., promoter, donor nucleic acid, selection gene) have only plant origins (i.e., no non-plant origin components are used). In an aspect, a plant, plant cell, or plant genome provided is cisgenic. Cisgenic plants, plant cells, and plant genomes provided can lead to ready-to-use tobacco lines. In another aspect, a tobacco plant provided comprises no non-tobacco genetic material or sequences.

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

In an aspect, a tobacco plant provided comprises one or more pmt mutations and further comprises reduced expression or activity of one or more genes involved in nicotine biosynthesis or 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), quinolate phosphoribosyl transferase (QPT), and S-adenosyl-methionine synthetase (SAMS). Nicotine Synthase, which catalyzes the condensation step between a nicotinic acid derivative and methylpyrrolinium cation, has not been elucidated although two candidate genes (A622 and NBB1) have been proposed. See US 2007/0240728 A1 and US 2008/0120737A1. A622 encodes an isoflavone reductase-like protein. In addition, several transporters may be involved in the translocation of nicotine. A transporter gene, named MATE, has been cloned and characterized (Morita et al., PNAS 106:2447-52 (2009)).

In an aspect, a tobacco plant provided comprises one or more pmt mutations and further comprises a reduced level of mRNA, protein, or both of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1, compared to a control tobacco plant. In another aspect, a tobacco plants provided comprises one or more pmt mutations and further comprises a transgene directly suppressing the expression of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, a tobacco plant provided comprises one or more pmt mutations and further comprises a transgene or mutation suppressing the expression or activity of one or more genes encoding a product selected from the group consisting of MPO, QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1.

In an aspect, a tobacco plant provided is from a tobacco type selected from the group consisting of flue-cured tobacco, air-cured tobacco, dark air-cured tobacco, dark fire-cured tobacco, Galpao tobacco, and Oriental tobacco. In another aspect, a tobacco plant provided is from a tobacco type selected from the group consisting of Burley tobacco, Maryland tobacco, and dark tobacco.

In an aspect, a tobacco plant provided is in a flue-cured tobacco background or exhibits one or more flue-cured tobacco characteristic described here. Flue-cured tobaccos (also called Virginia or bright tobaccos) amount to approximately 40% of world tobacco production. Flue-cured tobaccos are often also referred to as “bright tobacco” because of the golden-yellow to deep-orange color it reaches during curing. Flue-cured tobaccos have a light, bright aroma and taste. Flue-cured tobaccos are generally high in sugar and low in oils. Major flue-cured tobacco growing countries are Argentina, Brazil, China, India, Tanzania and the U.S. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a flue-cured tobacco background selected from the group consisting of CC 13, CC 27, CC 33, CC 37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399, K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC 71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT 220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety essentially derived from any one of the foregoing varieties. In another aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a flue-cured tobacco background selected from the group consisting of Coker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K 149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF, NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC 2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713, Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51, Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108, Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va 116, Va 182, and any variety essentially derived from any one of the foregoing varieties. See WO 2004/041006 A1. In a further aspect, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or lines are in any flue cured background selected from the group consisting of K326, K346, and NC196.

In an aspect, a tobacco plant provided is in an air-cured tobacco background or exhibits one or more air-cured tobacco characteristic described here. Air-cured tobaccos include Burley, Maryland, and dark tobaccos. The common factor is that curing is primarily without artificial sources of heat and humidity. Burley tobaccos are light to dark brown in color, high in oil, and low in sugar. Burley tobaccos are air-cured in barns. Major Burley growing countries are Argentina, Brazil, Italy, Malawi, and the U.S. Maryland tobaccos are extremely fluffy, have good burning properties, low nicotine and a neutral aroma. Major Maryland growing countries include the U.S. and Italy. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a Burley tobacco background selected from the group consisting of Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712, NCBH 129, Bu 21×Ky 10, HB04P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561, Pf561 and Va 509. In a further aspect, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or lines are in any Burley background selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488. In another aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a Maryland tobacco background selected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md 872 and Md 341.

In an aspect, a tobacco plant provided is in a dark air-cured tobacco background or exhibits one or more dark air-cured tobacco characteristic described here. Dark air-cured tobaccos are distinguished from other types primarily by its curing process which gives dark air-cured tobacco its medium- to dark-brown color and distinct aroma. Dark air-cured tobaccos are mainly used in the production of chewing tobacco and snuff. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in 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.

In an aspect, a tobacco plant provided is in a dark fire-cured tobacco background or exhibits one or more dark fire-cured tobacco characteristic described here. Dark fire-cured tobaccos are generally cured with low-burning wood fires on the floors of closed curing barns. Their leaves have low sugar content but high nicotine content. Dark fire-cured tobaccos are used for making pipe blends, cigarettes, chewing tobacco, snuff and strong-tasting cigars. Major growing regions for dark fire-cured tobaccos are Tennessee, Kentucky, and Virginia, USA. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in a dark fire-cured tobacco background selected from the group consisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole, Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, Small Stalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TN D94, TN D950, VA 309, and VA 359.

In an aspect, a tobacco plant provided is in an Oriental tobacco background or exhibits one or more Oriental tobacco characteristic described here. Oriental tobaccos are also referred to as Greek, aroma and Turkish tobaccos due to the fact that they are typically grown in eastern Mediterranean regions such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leaf size, characteristic of today's Oriental varieties, as well as its unique aroma properties are a result of the plant's adaptation to the poor soil and stressful climatic conditions in which it develop over many past centuries. In an aspect, a low-alkaloid or low-nicotine tobacco plant or seed provided is in an Oriental tobacco background selected from the group consisting of Izmir, Katerini, Samsun, Basma and Krumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne, Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra, Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any variety essentially derived from any one of the foregoing varieties.

In an aspect, low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties, or lines are essentially derived from or in the genetic background of BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or any commercial tobacco variety according to standard tobacco breeding techniques known in the art.

All foregoing mentioned specific varieties of dark air-cured, Burley, Maryland, dark fire-cured, or Oriental type are listed only for exemplary purposes. Any additional dark air-cured, Burley, Maryland, dark fire-cured, Oriental varieties are also contemplated in the present application.

Also provided are populations of tobacco plants described. In an aspect, a population of tobacco plants has a planting density of between about 5,000 and about 8000, between about 5,000 and about 7,600, between about 5,000 and about 7,200, between about 5,000 and about 6,800, between about 5,000 and about 6,400, between about 5,000 and about 6,000, between about 5,000 and about 5,600, between about 5,000 and about 5,200, between about 5,200 and about 8,000, between about 5,600 and about 8,000, between about 6,000 and about 8,000, between about 6,400 and about 8,000, between about 6,800 and about 8,000, between about 7,200 and about 8,000, or between about 7,600 and about 8,000 plants per acre. In another aspect, a population of tobacco plants is in a soil type with low to medium fertility.

Also provided are containers of seeds from tobacco plants described. A container of tobacco seeds of the present disclosure may contain any number, weight, or volume of seeds. For example, a container can contain at least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds. Alternatively, the container can contain at least, or greater than, about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seeds. Containers of tobacco seeds may be any container available in the art. By way of non-limiting example, a container may be a box, a bag, a packet, a pouch, a tape roll, a tube, or a bottle.

Also provided is cured tobacco material made from a low-alkaloid or low-nicotine tobacco plant described. Further provided is cured tobacco material made from a tobacco plant described with higher levels of total alkaloid or nicotine.

“Curing” is the aging process that reduces moisture and brings about the destruction of chlorophyll giving tobacco leaves a golden color and by which starch is converted to sugar. Cured tobacco therefore has a higher reducing sugar content and a lower starch content compared to harvested green leaf. In an aspect, green leaf tobacco provided can be cured using conventional means, e.g., flue-cured, barn-cured, fire-cured, air-cured or sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford) for a description of different types of curing methods. Cured tobacco is usually aged in a wooden drum (e.g., a hogshead) or cardboard cartons in compressed conditions for several years (e.g., two to five years), at a moisture content ranging from 10% to about 25%. See, U.S. Pat. Nos. 4,516,590 and 5,372,149. Cured and aged tobacco then can be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation. Fermentation typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of 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). Cure, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. In an aspect, the cured tobacco material of the present disclosure is sun-cured. In another aspect, the cured tobacco material of the present disclosure is flue-cured, air-cured, or fire-cured.

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

Tobacco plants, varieties, and lines provided herein comprising a mutant allele 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 as compared to the low alkaloid tobacco variety LA Burley 21 (LA BU 21). Similarly, tobacco plants, varieties, and lines provided herein comprising an RNAi construct that downregulates 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 as compared to the low alkaloid tobacco variety LA Burley 21 (LA BU 21).

LA BU 21 is a low total alkaloid tobacco line produced by incorporation of a low alkaloid gene(s) from a Cuban cigar variety into Burley 21 through several backcrosses (Legg et al., Crop Science, 10:212 (1970)), it has approximately 0.2% total alkaloids (dry weight) compared to the about 15% (dry weight) of its parent, Burley 21. LA B 21 has a leaf grade well below commercially acceptable standards.

In an aspect, a cured tobacco leaf comprising a mutant allele of pmt1a comprises no observable mold infection. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt1b comprises no observable mold infection. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt2 comprises no observable mold infection. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt3 comprises no observable mold infection. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt4 comprises no observable mold infection. In another aspect, a cured tobacco leaf 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 comprises no observable mold infection.

In an aspect, a cured tobacco leaf comprising a mutant allele of pmt1a comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt1b comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt2 comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt3 comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21. In another aspect, a cured tobacco leaf comprising a mutant allele of pmt4 comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21. In another aspect, a cured tobacco leaf 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 comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or line provided in any one of Tables 4A to 4E, Table 10, or Table 14 comprises no observable mold infection. In another aspect, a cured leaf from a tobacco plant, variety, or line provided in any one of Tables 4A to 4E, Table 10, or Table 14 comprises a reduced mold infection as compared to a control cured tobacco leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E comprises no observable mold infection. In another aspect, a cured leaf from a tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E comprises a reduced mold infection as compared to a control cured leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or line comprising a mutant allele of pmt1a comprises a higher leaf grade than a control cured leaf from the variety LA BU 21. In an aspect, a cured leaf from a tobacco plant, variety, or line comprising a mutant allele of pmt1b comprises a higher leaf grade than a control cured leaf from the variety LA BU 21. In an aspect, a cured leaf from a tobacco plant, variety, or line comprising a mutant allele of pmt2 comprises a higher leaf grade than a control cured leaf from the variety LA BU 21. In an aspect, a cured leaf from a tobacco plant, variety, or line comprising a mutant allele of pmt3 comprises a higher leaf grade than a control cured leaf from the variety LA BU 21. In an aspect, a cured leaf from a tobacco plant, variety, or line comprising a mutant allele of pmt4 comprises a higher leaf grade than a control cured leaf from the variety LA BU 21. In another aspect, a cured tobacco leaf from a plant, variety, or line 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 comprises a higher leaf grade than a control cured leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or line provided in any one of Tables 4A to 4E, Table 10, or Table 14 comprises a higher leaf grade than a control cured leaf from the variety LA BU 21.

In an aspect, a cured leaf from a tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E comprises a higher leaf grade than a control cured leaf from the variety LA BU 21.

In an aspect, a “reduced mold infection” refers to a reduced area of infected leaf. In another aspect, a “reduced mold infection” refers to a reduced number of viable mold spores on an infected leaf. Standard methods of detecting and counting viable mold spores are known and available in the art.

In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 1% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 2% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 3% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 4% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 5% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 10% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 15% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 20% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 25% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 30% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 35% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 40% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 50% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 60% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 70% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 75% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 80% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 90% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of at least 95% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of 100% as compared to a control leaf.

In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 90% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 80% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 70% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 60% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 50% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 40% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 30% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 20% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 1% and 10% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 10% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 20% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 30% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 40% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 50% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 60% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 70% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 80% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 90% and 100% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 10% and 75% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 25% and 75% as compared to a control leaf. In an aspect, a reduced mold infection comprises a reduction of infected leaf area of between 25% and 50% as compared to a control leaf.

In an aspect, mold infecting, cured tobacco is of a genus selected from the group consisting of Cladosporium, Alternaria, Aspergillus, and Mucor.

Tobacco material obtained from the tobacco lines, 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 or derived from tobacco that is intended for human use or consumption.

Tobacco products provided include, without limitation, cigarette products (e.g., cigarettes and bidi cigarettes), cigar products (e.g., cigar wrapping tobacco and cigarillos), pipe tobacco products, products derived from tobacco, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tabs, shaped parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, and the like. See, e.g., U.S. Patent Publication No. US 2006/0191548.

As used herein, “cigarette” refers a tobacco product having a “rod” and “filler”. The cigarette “rod” includes the cigarette paper, filter, plug wrap (used to contain filtration materials), tipping paper that holds the cigarette paper (including the filler) to the filter, and all glues that hold these components together. The “filler” includes (1) all tobaccos, including but not limited to reconstituted and expanded tobacco, (2) non-tobacco substitutes (including but not limited to herbs, non-tobacco plant materials and other spices that may accompany tobaccos rolled within the cigarette paper), (3) casings, (4) flavorings, and (5) all other additives (that are mixed into tobaccos and substitutes and rolled into the cigarette).

As used herein, “reconstituted tobacco” refers to a part of tobacco filler made from tobacco dust and other tobacco scrap material, processed into sheet form and cut into strips to resemble tobacco. In addition to the cost savings, reconstituted tobacco is very important for its contribution to cigarette taste from processing flavor development using reactions between ammonia and sugars.

As used herein, “expanded tobacco” refers to a part of tobacco filler which is processed through expansion of suitable gases so that the tobacco is “puffed” resulting in reduced density and greater filling capacity. It reduces the weight of tobacco used in cigarettes.

Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, where the rod of smokable material includes cured tobacco within a tobacco blend. In an aspect, a tobacco product of the present disclosure is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco. In another aspect, a tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not combusted and include, but not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff Chewing tobacco is coarsely divided tobacco leaf that is typically packaged in a large pouch-like package and used in a plug or twist. Moist smokeless tobacco is a moist, more finely divided tobacco that is provided in loose form or in pouch form and is typically packaged in round cans and used as a pinch or in a pouch placed between an adult tobacco consumer's cheek and gum. Snus is a heat treated smokeless tobacco. Dry snuff is finely ground tobacco that is placed in the mouth or used nasally. In a further aspect, a 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, a tobacco product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an e-cigarette, an electronic vaporing device.

In an aspect, a tobacco product of the present disclosure can be a blended tobacco product. In another aspect, a tobacco product of the present disclosure can be a low nicotine tobacco product. In a further aspect, a tobacco product of the present disclosure may comprise nornicotine at a level of less than about 3 mg/g. For example, the nornicotine content in such a product 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.

In an aspect, cured tobacco material or tobacco products provided comprise an average nicotine or total alkaloid level selected from the group consisting of 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% on a dry weight basis. In another aspect, cured tobacco material or tobacco products provided comprise an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.02%, between 0.02% and 0.05%, between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%, between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%, between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%, between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%, between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between 1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between 1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between 1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1% and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and 2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and 2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%, between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%, between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis. In a further aspect, cured tobacco material or tobacco products provided comprise an average nicotine or total alkaloid level selected from the group consisting of about between 0.01% and 0.1%, between 0.02% and 0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%, between 0.05% and 0.5%, between 0.75% and 1%, between 0.1% and 1.5%, between 0.15% and 2%, between 0.2% and 3%, and between 0.3% and 3.5% on a dry weight basis.

The present disclosure also provides methods for breeding tobacco lines, cultivars, or varieties comprising a desirable level of total alkaloid or nicotine, e.g., low nicotine or nicotine free. Breeding can be carried out via any known procedures. DNA fingerprinting, SNP mapping, haplotype mapping or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed a desirable trait or allele into a tobacco plant. For example, a breeder can create segregating populations in a F₂ or backcross generation using F1 hybrid plants or further crossing the F1 hybrid plants with other donor plants with an agronomically desirable genotype. Plants in the F₂ or backcross generations can be screened for a desired agronomic trait or a desirable chemical profile using one of the techniques known in the art or listed herein. Depending on the expected inheritance pattern or the MAS technology used, self-pollination of selected plants before each cycle of backcrossing to aid identification of the desired individual plants can be performed. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered. A recurrent parent in the present disclosure can be a flue-cured variety, a Burley variety, a dark air-cured variety, a dark fire-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 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y., incorporated herein by reference in their entirety.

Results of a plant breeding program using the tobacco plants described includes useful lines, cultivars, varieties, progeny, inbreds, and hybrids of the present disclosure. As used herein, the term “variety” refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A “pure line” variety may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A variety can be essentially derived from another line or variety. As defined by the International Convention for the Protection of New Varieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10, 1972; on Oct. 23, 1978; and on Mar. 19, 1991), a variety is “essentially derived” from an initial variety if: a) it is predominantly derived from the initial variety, or from a variety that is predominantly derived from the initial variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety; b) it is clearly distinguishable from the initial variety; and c) except for the differences which result from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety. Essentially derived varieties can be obtained, for example, by the selection of a natural or induced mutant, a somaclonal variant, a variant individual from plants of the initial variety, backcrossing, or transformation. A first tobacco variety and a second tobacco variety from which the first variety is essentially derived, are considered as having essentially identical genetic background. A “line” as distinguished from a variety most often denotes a group of plants used non-commercially, for example in plant research. A line typically displays little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits.

In an aspect, this disclosure provides a tobacco plant, variety, line, or cell comprising one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E.

In another aspect, this disclosure provides a tobacco plant, variety, line, or cell derived from any tobacco plant, variety, or line provided in any one of Tables 4A to 4E, Table 10, or Table 14.

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

In an aspect, the present disclosure provides a method of introgressing a low nicotine trait into a tobacco variety, the method comprising: (a) crossing a first tobacco variety comprising a low nicotine trait with a second tobacco variety without 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 to 4E, Tables 5A to 5E, Table 10, and Tables 12A to 12E; and (c) selecting a progeny tobacco plant comprising the pmt mutant allele. In another aspect, these methods further comprise backcrossing the selected progeny tobacco plant with the second tobacco variety. In a further aspect, these methods further comprise: (d) crossing the selected progeny plant with itself or with the second tobacco variety to produce one or more further progeny tobacco plants; and (e) selecting a further progeny tobacco plant comprising a low nicotine trait. In an aspect, the step (e) of selecting comprises marker-assisted selection. In an aspect, these methods produce a single gene conversion comprising a low nicotine trait. In an aspect, these methods produce a single gene conversion comprising a pmt mutant allele. In an aspect, the second tobacco variety is an elite variety. In another aspect, the genotyping step of these methods involve one or more molecular marker assays. In another aspect, the genotyping may involve a polymorphic marker comprising a polymorphism selected from the group consisting of single nucleotide polymorphisms (SNPs), insertions or deletions in DNA sequence (Indels), simple sequence repeats of DNA sequence (SSRs), a restriction fragment length polymorphism (RFLP), and a tag SNP.

As used herein, “locus” is a chromosomal locus or region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. A “locus” can be shared by two homologous chromosomes to refer to their corresponding locus or region. As used herein, “allele” refers to an alternative nucleic acid sequence of a gene or at a particular locus (e.g., a nucleic acid sequence of a gene or locus that is different than other alleles for the same gene or locus). Such an allele 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 relative to the wild-type allele. A mutant allele for a gene may have a reduced or eliminated activity or expression level for the gene relative 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, then the plant is described as being heterozygous for the mutant allele. However, if both alleles at a locus are mutant alleles, then the plant is described as being homozygous for the mutant alleles. A plant homozygous for mutant alleles at a locus may comprise the same mutant allele or different mutant alleles if heteroallelic or biallelic.

As used herein, “introgression” or “introgress” refers to the transmission of a desired allele of a genetic locus from one genetic background to another.

As used herein, “crossed” or “cross” means to produce progeny via fertilization (e.g. cells, seeds or plants) and includes crosses between plants (sexual) and self-fertilization (selfing).

As used herein, “backcross” and “backcrossing” refer to the process whereby a progeny plant is repeatedly crossed back to one of its parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. The initial cross gives rise to 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 an aspect, a backcross is performed repeatedly, with a progeny individual of each successive backcross generation being itself backcrossed to the same parental genotype.

As used herein, “single gene converted” or “single gene conversion” refers to plants that are developed using a plant breeding technique known as backcrossing, or via genetic engineering, where essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single gene transferred into the variety via the backcrossing technique or via genetic engineering.

As used herein, “elite variety” means any variety that has resulted from breeding and selection for superior agronomic performance.

As used herein, “selecting” or “selection” in the context of marker-assisted selection or breeding refer to the act of picking or choosing desired individuals, normally from a population, based on certain pre-determined criteria.

As used herein, the term “trait” refers to one or more detectable characteristics of a cell or organism which can be influenced by genotype. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, genomic analysis, an assay for a particular disease tolerance, etc. In some cases, a phenotype is directly controlled by a single gene or genetic locus, e.g., a “single gene trait.” In other cases, a phenotype is the result of several genes.

As used herein, “marker assay” means a method for detecting a polymorphism at a particular locus using a particular method, e.g., 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, allelic specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based technologies, and nucleic acid sequencing technologies, etc.

As used herein, “marker assisted selection” (MAS) is a process by which phenotypes are selected based on marker genotypes. “Marker assisted selection breeding” refers to the process of selecting a desired trait or traits in a plant or plants by detecting one or more nucleic acids from the plant, where the nucleic acid is linked to the desired trait, and then selecting the plant or germplasm possessing those one or more nucleic acids.

As used herein, “polymorphism” means the presence of one or more variations in a population. A polymorphism may manifest as a variation in the nucleotide sequence of a nucleic acid or as a variation in the amino acid sequence of a protein. Polymorphisms include the presence of one or more variations of a nucleic acid sequence or nucleic acid feature at one or more loci in a population of one or more individuals. The variation may comprise but is not limited to one or more nucleotide base changes, the insertion of one or more nucleotides or the deletion of one or more nucleotides. A polymorphism may arise from random processes in nucleic acid replication, through mutagenesis, as a result of mobile genomic elements, from copy number variation and during the process of meiosis, such as unequal crossing over, genome duplication and chromosome breaks and fusions. The variation can be commonly found or may exist at low frequency within a population, the former having greater utility in general plant breeding and the latter may be associated with rare but important phenotypic variation. Useful polymorphisms may include single nucleotide polymorphisms (SNPs), insertions or deletions in DNA sequence (Indels), simple sequence repeats of DNA sequence (SSRs), a restriction fragment length polymorphism (RFLP), and a tag SNP. A genetic marker, a gene, a DNA-derived sequence, a RNA-derived sequence, a promoter, a 5′ untranslated region of a gene, a 3′ untranslated region of a gene, microRNA, siRNA, a tolerance locus, a satellite marker, a transgene, mRNA, ds mRNA, a transcriptional profile, and a methylation pattern may also comprise polymorphisms. In addition, the presence, absence, or variation in copy number of the preceding may comprise polymorphisms.

As used herein, “SNP” or “single nucleotide polymorphism” means a sequence variation that occurs when a single nucleotide (A, T, C, or G) in the genome sequence is altered or variable. “SNP markers” exist when SNPs are mapped to sites on the genome.

As used herein, “marker” or “molecular marker” or “marker locus” is a term used to denote 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 so long as it is inherited differentially and exhibits linkage disequilibrium with a phenotypic trait of interest. Each marker is therefore an indicator of a specific segment of DNA, having a unique nucleotide sequence. The map positions provide a measure of the relative positions of particular markers with respect to one another. When a trait is stated to be linked to a given marker it will be understood that the actual DNA segment whose sequence affects the trait generally co-segregates with the marker. More precise and definite localization of a trait can be obtained if markers are identified on both sides of the trait. By measuring the appearance of the marker(s) in progeny of crosses, the existence of the trait can be detected by relatively simple molecular tests without actually evaluating the appearance of the trait itself, which can be difficult and time-consuming because the actual evaluation of the trait requires growing plants to a stage and/or under environmental conditions where the trait can be expressed.

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 a technique known in the art. Without limitation, an example of a desired trait is herbicide resistance, pest resistance, disease resistance; high yield; high grade index value; curability; curing quality; mechanical harvestability; holding ability; leaf quality; height, plant maturation (e.g., early maturing, early to medium maturing, medium maturing, medium to late maturing, or late maturing); stalk size (e.g., a small, medium, or a large stalk); or leaf number per plant (e.g., a small (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) number of leaves), or any combination. In an aspect, low-nicotine or nicotine-free tobacco plants or seeds disclosed comprise one or more transgenes expressing one or more insecticidal proteins, such as, for example, a crystal protein of Bacillus thuringiensis or a vegetative insecticidal protein from Bacillus cereus, such as VIP3 (see, for example, Estruch et al. (1997) Nat. Biotechnol. 15:137). In another aspect, tobacco plants further comprise an introgressed trait conferring resistance to brown stem rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S. Pat. No. 5,491,081).

The present disclosure also provides pmt mutant tobacco plants comprising an altered nicotine or total alkaloid level but having a yield comparable to the yield of corresponding initial tobacco plants without such a nicotine level alternation. In an aspect, a pmt mutant variety provides a yield selected from the group consisting of about between 1200 and 3500, between 1300 and 3400, between 1400 and 3300, between 1500 and 3200, between 1600 and 3100, between 1700 and 3000, between 1800 and 2900, between 1900 and 2800, between 2000 and 2700, between 2100 and 2600, between 2200 and 2500, and between 2300 and 2400 lbs/acre. In another aspect, a pmt mutant tobacco variety provides a yield selected from the group consisting of about between 1200 and 3500, between 1300 and 3500, between 1400 and 3500, between 1500 and 3500, between 1600 and 3500, between 1700 and 3500, between 1800 and 3500, between 1900 and 3500, between 2000 and 3500, between 2100 and 3500, between 2200 and 3500, between 2300 and 3500, between 2400 and 3500, between 2500 and 3500, between 2600 and 3500, between 2700 and 3500, between 2800 and 3500, between 2900 and 3500, between 3000 and 3500, and between 3100 and 3500 lbs/acre. In a further aspect, pmt mutant tobacco plants provide a yield between 65% and 130%, between 70% and 130%, between 75% and 130%, between 80% and 130%, between 85% and 130%, between 90% and 130%, between 95% and 130%, between 100% and 130%, between 105% and 130%, between 110% and 130%, between 115% and 130%, or between 120% and 130% of the yield of a control plant having essentially identical genetic background except for pmt mutation(s). In a further aspect, pmt mutant tobacco plants provide a yield between 70% and 125%, between 75% and 120%, between 80% and 115%, between 85% and 110%, or between 90% and 100% of the yield of a control plant having essentially identical genetic background except for pmt mutations.

In an aspect, a tobacco plant disclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobacco variety) comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) without substantially impacting a trait selected from the group consisting of yield, ripening and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll level, mesophyll cell number per unit leaf area, and end-product quality after curing.

In an aspect, a tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a trait substantially comparable to an unmodified control plant, where the trait is selected from the group consisting of yield, ripening and senescence, susceptibility to insect herbivory, polyamine content after topping, chlorophyll level, mesophyll cell number per unit leaf area, and end-product quality after curing.

In an aspect, a tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a yield which is more than 80%, more than 85%, more than 90%, more than 95%, more than 100%, more than 105%, more than 110%, more than 115%, more than 120%, more than 125%, more than 130%, more than 135%, or more than 140% relative to the yield of an unmodified control plant. In an aspect, a tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a yield which is between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 115%, or between 100% and 110% relative to the yield of an unmodified control plant. In an aspect, a tobacco plant disclosed comprises a modification conferring a desired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) and further comprises a yield which is between 70% and 80%, between 75% and 85%, between 80% and 90%, between 85% and 95%, between 90% and 100%, between 95% and 105%, between 105% and 115%, between 110% and 120%, between 115% to 125%, between 120% and 130%, between 125 and 135%, or between 130% and 140% relative to the yield of an unmodified control plant.

In an aspect, a low-nicotine or nicotine-free tobacco variety disclosed is adapted for machine harvesting. In another aspect, a low-nicotine or nicotine-free tobacco variety disclosed is harvested mechanically.

In an aspect, tobacco plants provided are hybrid plants. Hybrids can be produced by preventing self-pollination of female parent plants (e.g., seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing F1 hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be produced by male sterility (MS), or transgenic male sterility where a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. Female parent plants containing MS are particularly useful. In aspects in which the female parent plants are MS, pollen may be harvested from male fertile plants and applied manually to the stigmas of MS female parent plants, and the resulting F1 seed is harvested.

Plants can be used to form single-cross tobacco F1 hybrids. Pollen from a male parent plant is manually transferred to an emasculated female parent plant or a female parent plant that is male sterile to form F1 seed. Alternatively, three-way crosses can be carried out where a single-cross F1 hybrid is used as a female parent and is crossed with a different male parent. As another alternative, double-cross hybrids can be created where the F1 progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid.

In an aspect, a low-nicotine or nicotine-free tobacco variety is male sterile. In another aspect, a low-nicotine or nicotine-free tobacco variety is cytoplasmic male sterile. Male sterile tobacco plants may be produced by any method known in the art. Methods of producing male sterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.

In an aspect, this disclosure provides a male sterile tobacco plant, variety, or line comprising one or more pmt mutations provided in any one of Tables 5A to 5E and Tables 12A to 12E.

In another aspect, this 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 to 4E, Table 10, or Table 14.

In an aspect, this disclosure provides the male sterile line dCS11. In another aspect, this disclosure provides the male sterile line dCS12. In another aspect, this disclosure provides the male sterile line dCS13. In another aspect, this disclosure provides the male sterile line dCS14. In another aspect, this disclosure provides the male sterile line dCS15. In another aspect, this disclosure provides the male sterile line dCS16. In another aspect, this disclosure provides the male sterile line dCS17. In another aspect, this disclosure provides the male sterile line dCS18. In another aspect, this disclosure provides the male sterile line dS697.

In a further aspect, tobacco parts provided include, but are not limited to, a leaf, a stem, a root, a seed, a flower, pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a callus, a tissue culture, a shoot, a cell, and a protoplast. In an aspect, tobacco part provided does not include seed. In an aspect, this disclosure provides tobacco plant cells, tissues, and organs that are not reproductive material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides tobacco plant cells, tissues, and organs that are reproductive material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides tobacco plant cells, tissues, and organs that cannot maintain themselves via photosynthesis. In another aspect, this disclosure provides somatic tobacco plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction.

Cells, tissues and organs can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot, stem, pod, flower, infloresence, stalk, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem, vascular tissue. In another aspect, this disclosure provides a tobacco plant chloroplast. In a further aspect, this disclosure provides epidermal cells, stomata cell, leaf or root hairs, a storage root, or a tuber. In another aspect, this disclosure provides a tobacco protoplast.

Skilled artisans understand that tobacco plants naturally reproduce via seeds, not via asexual reproduction or vegetative propagation. In an aspect, this disclosure provides tobacco endosperm. In another aspect, this disclosure provides tobacco endosperm cells. In a further aspect, this disclosure provides a male or female sterile tobacco plant, which cannot reproduce without human intervention.

In an aspect, the present 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 to 10, and fragments thereof. In an aspect, the present 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 to 15.

As used herein, the term “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

The present disclosure further provides a method manufacturing a tobacco product comprising tobacco material from tobacco plants disclosed. In an aspect, methods comprise conditioning aged tobacco material made from tobacco plants to increase its moisture content from between about 12.5% and about 13.5% to about 21%, blending the conditioned tobacco material to produce a desirable blend. In an aspect, the method of manufacturing a tobacco product further comprises casing or flavoring the blend. Generally, during the casing process, casing or sauce materials are added to blends to enhance their quality by balancing the chemical composition and to develop certain desired flavor characteristics. Further details for the casing process can be found in Tobacco Production, Chemistry and Technology, Edited by L. Davis and M. Nielsen, Blackwell Science, 1999.

Tobacco material provided can be also 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 fire cured, burley, flue cured, and cigar filler or wrapper, as well as the products from the whole leaf stemming operation. In an aspect, tobacco fibers include up to 70% dark tobacco on a fresh weight basis. For example, tobacco can be conditioned by heating, sweating and/or pasteurizing steps as described in U.S. Publication Nos. 2004/0118422 or 2005/0178398.

Tobacco material provided can be subject to fermentation. Fermenting typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of dry weight. See, e.g., U.S. Pat. Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition to modifying the aroma of the leaf, fermentation can change either or both the color and texture of a leaf. Also during the fermentation process, evolution gases can be produced, oxygen can be taken up, the pH can change, and the amount of water retained can change. See, for example, U.S. Publication No. 2005/0178398 and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, or cured and fermented tobacco can be further processed (e.g., cut, expanded, blended, milled or comminuted) prior to incorporation into the oral product. The tobacco, in some cases, is long cut fermented cured moist tobacco having an oven volatiles content of between 48 and 50 weight percent prior to mixing with the copolymer and optionally flavorants and other additives.

In an aspect, tobacco material provided can be processed to a desired size. In an aspect, tobacco fibers can be processed to have an average fiber size of less than 200 micrometers. In an aspect, tobacco fibers are between 75 and 125 micrometers. In another aspect, tobacco fibers are processed to have a size of 75 micrometers or less. In an aspect, tobacco fibers include long cut tobacco, which can be cut or shredded into widths of about 10 cuts/inch up to about 110 cuts/inch and lengths of about 0.1 inches up to about 1 inch. Double cut tobacco fibers can have a range of particle sizes such that about 70% of the double cut tobacco fibers falls between the mesh sizes of −20 mesh and 80 mesh.

Tobacco material provided can be processed to have a total oven volatiles content of about 10% by weight or greater; about 20% by weight or greater; about 40% by weight or greater; about 15% by weight to about 25% by weight; about 20% by weight to about 30% by weight; about 30% by weight to about 50% by weight; about 45% by weight to about 65% by weight; or about 50% by weight to about 60% by weight. Those of skill in the art will appreciate that “moist” tobacco typically refers to tobacco that has an oven volatiles content of between about 40% by weight and about 60% by weight (e.g., about 45% by weight to about 55% by weight, or about 50% by weight). As used herein, “oven volatiles” are determined by calculating the percentage of weight loss for a sample after drying the sample in a pre-warmed forced draft oven at 110° C. for 3.25 hours. The oral product can have a different overall oven volatiles content than the oven volatiles content of the tobacco fibers used to make the oral product. The processing steps described can reduce or increase the oven volatiles content.

Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified.

EXAMPLES

Example 1: Expression Profiling of Five PMT Genes

Nicotine biosynthesis starts with conversion of polyamine putrescine to N-methylputrescine by the enzyme putrescine N-methyl transferase (PMT). This is a step that commits precursor metabolites to nicotine biosynthesis. Genes encoding PMT (PMT1a, PMT1b, PMT2, PMT3 and PMT4) are present in the tobacco (Nicotiana tabacum) genome. Table 1A lists genomic DNA sequences, cDNA sequences, and protein sequences of five PMT genes. Tables 1B and 1C provide sequence identities among five PMT genes. Pooled expression levels from before topping to harvest provide support that, without being limited by any particular theory, PMT1a and PMT3 represent two major PMT genes (FIG. 1).

TABLE 1A
Sequences of five tobacco PMT genes.

	Genomic DNA Sequence
	(including regions such as	cDNA	Protein
	promoter, 5′ UTR, introns,	Sequence	Sequence
Gene	3′ UTR, and terminator)	(SEQ ID	(SEQ ID
Name	(SEQ ID No.)	No.)	No.)

PMT1b	1	6	11
PMT1a	2	7	12
PMT2	3	8	13
PMT3	4	9	14
PMT4	5	10	15

TABLE 1B
cDNA sequence identity among five tobacco
PMT genes determined by Clustal2.1.

cDNA %
identity	PMT1a	PMT1b	PMT2	PMT3	PMT4

PMT1a	100
PMT1b	98.85	100
PMT2	91.81	91.71	100
PMT3	93.71	93.53	91.79	100
PMT4	94.24	94.06	92.75	94.59	100

TABLE 1C
Protein sequence identity among five tobacco
PMT genes determined by Clustal2.1.

Protein %
identity	PMT1a	PMT1b	PMT2	PMT3	PMT4

PMT1a	100
PMT1b	98.4	100
PMT2	95.42	95.75	100
PMT3	97.48	97.76	96.23	100
PMT4	96.27	96.8	96.32	97.63	100

TABLE 1D
PMT1b genomic sequence (SEQ ID No. 1) annotation.

	Element	location
	5′ sequence	1 . . . 1000
	exon 1	1001 . . . 1292
	intron 1	1293 . . . 1464
	exon 2	1465 . . . 1541
	intron 2	1542 . . . 1623
	exon 3	1624 . . . 1851
	intron 3	1852 . . . 1971
	exon 4	1972 . . . 2044
	intron 4	2045 . . . 2143
	exon 5	2144 . . . 2215
	intron 5	2216 . . . 2333
	exon 6	2334 . . . 2529
	intron 6	2530 . . . 3033
	exon 7	3034 . . . 3166
	intron 7	3167 . . . 3260
	exon 8	3261 . . . 3317
	3′ sequence	3318 . . . 4317

TABLE 1E
PMT1b genomic sequence (SEQ ID No. 2) annotation.

	Element	location
	5′ sequence	1 . . . 1000
	exon 1	1001 . . . 1294
	intron 1	1295 . . . 1422
	exon 2	1423 . . . 1497
	intron 2	1498 . . . 1579
	exon 3	1580 . . . 1810
	intron 3	1811 . . . 1932
	exon 4	1933 . . . 2003
	intron 4	2004 . . . 2102
	exon 5	2103 . . . 2175
	intron 5	2176 . . . 2293
	exon 6	2294 . . . 2487
	intron 6	2488 . . . 2925
	exon 7	2926 . . . 3058
	intron 7	3059 . . . 3153
	exon 8	3154 . . . 3210
	3′ sequence	3211 . . . 4210

TABLE 1F
PMT2 genomic sequence (SEQ ID No. 3) annotation.

	Element	location
	5′ sequence	1 . . . 792
	exon 1	793 . . . 1020
	intron 1	1021 . . . 1201
	exon 2	1202 . . . 1276
	intron 2	1277 . . . 1358
	exon 3	1359 . . . 1589
	intron 3	1590 . . . 1694
	exon 4	1695 . . . 1765
	intron 4	1766 . . . 1875
	exon 5	1876 . . . 1948
	intron 5	1949 . . . 2037
	exon 6	2038 . . . 2231
	intron 6	2232 . . . 2397
	exon 7	2398 . . . 2530
	intron 7	2531 . . . 2629
	exon 8	2630 . . . 2686
	3′ sequence	2687 . . . 3686

TABLE 1G
PMT3 genomic sequence (SEQ ID No. 4) annotation.

	Element	location
	5′ sequence	1 . . . 1000
	exon 1	1001 . . . 1312
	intron 1	1313 . . . 1562
	exon 2	1563 . . . 1637
	intron 2	1638 . . . 1731
	exon 3	1732 . . . 1962
	intron 3	1963 . . . 2050
	exon 4	2051 . . . 2121
	intron 4	2122 . . . 2230
	exon 5	2231 . . . 2303
	intron 5	2304 . . . 2397
	exon 6	2398 . . . 2591
	intron 6	2592 . . . 2750
	exon 7	2751 . . . 2883
	intron 7	2884 . . . 2978
	exon 8	2979 . . . 3035
	3′ sequence	3036 . . . 4035

TABLE 1H
PMT4 genomic sequence (SEQ ID No. 5) annotation.

	Element	location
	5′ sequence	1 . . . 1000
	exon 1	1001 . . . 1426
	intron 1	1427 . . . 1609
	exon 2	1610 . . . 1684
	intron 2	1685 . . . 1766
	exon 3	1767 . . . 1997
	intron 3	1998 . . . 2112
	exon 4	2113 . . . 2183
	intron 4	2184 . . . 2290
	exon 5	2291 . . . 2363
	intron 5	2364 . . . 2452
	exon 6	2453 . . . 2646
	intron 6	2647 . . . 3146
	exon 7	3147 . . . 3279
	intron 7	3280 . . . 3374
	exon 8	3375 . . . 3431
	3′ sequence	3432 . . . 4431

Example 2: PMT Genome Editing and Tobacco Line Development

PMT knockout mutants are produced by editing various PMT genes. Tobacco protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding a genome editing technology 1 (GET 1) protein or a genome editing technology (GET) 2 protein and specific guide RNAs (gRNAs) targeting PMT genes at desired positions. Table 2 lists gRNA sequences used for PMT editing. Some gRNAs (e.g., Nos. 6 and 7) are pooled together for targeting multiple PMT genes in a single transfection.

Transfected protoplasts are then immobilized in 1% agarose bead and subjected to tissue culture. When calli grow up to ˜1 mm in diameter, they are spread on TOM2 plates. Calli are screened for insertions or deletions (indels) at the target positions using fragment analysis. Candidates, showing size shifts compared to wildtype control, are selected for further culture and the consequent shoots are tested by fragment analysis again to confirm the presence of indels. Rooted shoots are potted and sequenced for the target positions to determine the exact sequences deleted. Young leaf from each plant is harvested and PCR amplified for PMT fragments using phirekit. PMT Libraries for each line is indexed and 384 lines are pooled and sequenced using Miseq.

SNP analysis is carried out to determine both the exact edited pmt mutant allele sequences and the zygosity state at each PMT gene locus. Table 3 provides the zygosity information of representative edited plants. Tables 4A to 4E provide indels sequence information in each edited line of various tobacco varieties (e.g., K326, TN90, NLM, oriental). Tables 5A to 5E provide genomic sequences of about 40 nucleotides from each pmt mutant allele with the edited site in the middle of the genomic sequence (e.g., 20 nucleotides on each side of the deleted or inserted sequence site).

TABLE 2
gRNA sequences used in 2 genome editing technologies and their target genes. “Y”
represents that a gRNA targets that PMT gene, while “-” represents that a gRNA does not
target that PMT gene.

	Genome Editing
gRNA	(GET)		Target genes

No.	Technology	gRNA sequence	PMTlb	PMT1a	PMT2	PMT3	PMT4
1	GET 1	CCCATGAACGGCCACCAAAA	Y	Y	—	—	—
		(SEQ ID NO: 16)
2	GET 1	GGCACTTCCAAACACCAAAA	Y	Y	Y	Y	Y
		(SEQ ID NO: 17)
3	GET 1	GTTGTTCGGATGTCCCATTC	Y	Y	—	—	—
		(SEQ ID NO: 18)
4	GET 1	CTAAACTCTGAAAACCAACC	Y	—	Y	Y	—
		(SEQ ID NO: 19)
5	GET 1	TTTCAGAGTTTAGCGCATTA	Y	Y	Y	Y	Y
		(SEQ ID NO: 20)
6	GET 2	GATGGAGCAATTCAACATACAGA	Y	Y	—	—	—
		(SEQ ID NO: 21)
7	GET 2	GATGGAGCAATTCAACACACAGA	—	—	Y	Y	Y
		(SEQ ID NO: 22)

TABLE 3
Zygosity of individual PMT genic locus in selected pmt mutants
in various background produced by genome editing using GET2.

Variety	Line	PMT1b	PMT1a	PMT2	PMT3	PMT4
Basma	18GH203	1	2	2	2	1
Basma	18GH341	1	2	2	2	1
K326	17GH1678	2	2	1	1	2
K326	17GH1680	1	2	1	1	1
K326	17GH1804	1	2	1	1	1
K326	17GH1898	1	2	1	1	1
K326	18GH207	1	2	1	1	1
K326	18GH342	1	2	1	1	1
K326	18GH343	1	2	1	1	1
K326	18GH348	1	1	1	1	1
K326	18GH349	1	1	1	1	1
K326	18GH355	1	2	2	3	2
K326	18GH359	1	2	1	1	1
K326	18GH64	2	1	2	1	1
K326	18GH682	1	2	2	2	2
K326	18GH692	1	2	2	2	1
K326	18GH697	1	1	1	1	1
K326	18GH922	1	1	1	1	1
K326	18GH957	1	1	2	1	1
K326	17GH1808	1	2	1	2	1
K326	17GH1810	1	1	1	2	2
K326	17GH1886	—	—	2	1	—
K326	17GH1888	—	1	1	—	—
K326	17GH1889	—	1	1	—	—
K326	17GH1892	3	1	2	2	—
K326	17GH1893	1	1	1	1	1
K326	17GH1901	1	1	1	2	2
K326	17GH1902	1	1	1	2	2
K326	18GH3	—	1	1	1	—
Katerini	18GH125	2	2	1	2	1
Katerini	18GH208	2	1	1	2	1
Katerini	18GH403	1	1	1	1	—
Katerini	18GH414	2	1	1	1	1
Katerini	18GH434	2	1	1	2	1
Katerini	18GH436	2	1	1	4	1
Katerini	18GH437	1	2	1	2	2
Katerini	18GH449	2	2	1	1	1
Katerini	18GH706	2	1	1	2	1
Katerini	18GH709	2	2	1	1	1
Katerini	18GH710	1	1	2	2	1
Katerini	18GH716	2	2	1	2	2
Katerini	18GH729	1	1	2	1	1
Katerini	18GH731	1	1	1	2	1
Katerini	18GH752	2	1	1	1	1
Katerini	18GH756	1	1	1	2	2
Katerini	18GH768	1	1	1	1	2
Katerini	18GH771	1	1	2	2	1
Katerini	18GH776	2	2	1	1	2
Katerini	18GH800	2	2	1	1	2
Katerini	18GH818	1	1	1	2	—
NLMz	18GH10	1	1	1	1	1
NLMz	18GH1004	2	1	1	2	2
NLMz	18GH1033	2	1	1	2	2
NLMz	18GH132	1	2	2	3	1
NLMz	18GH134	1	2	1	1	1
NLMz	18GH217	1	2	2	1	2
NLMz	18GH456	2	1	1	1	1
NLMz	18GH457	1	1	1	1	1
NLMz	18GH460	1	2	3	1	1
NLMz	18GH465	2	1	1	2	2
NLMz	18GH71	1	1	1	1	1
NLMz	18GH830	1	1	1	1	1
NLMz	18GH831	1	2	1	—	1
NLMz	18GH836	1	1	1	1	1
NLMz	18GH841	2	2	1	—	1
NLMz	18GH974	2	1	2	2	1
NLMz	18GH981	1	1	2	2	2
NLMz	18GH994	2	1	1	2	2
NLMz	17GH1905	1	2	1	2	2
NLMz	18GH128	—	2	2	—	1
NLMz	18GH130	2	2	1	1	1
NLMz	18GH131	1	3	2	—	1
NLMz	18GH133	2	3	2	—	1
NLMz	18GH136	—	—	1	—	—
NLMz	18GH216	2	2	1	1	2
NLMz	18GH227	1	1	1	—	1
NLMz	18GH5	1	2	1	2	2
NLMz	18GH6	1	2	3	1	1
NLMz	18GH65	2	2	2	2	1
NLMz	18GH66	1	2	1	2	2
NLMz	18GH69	—	1	—	2	1
NLMz	18GH72	2	2	2	2	1
NLMz	18GH73	—	2	2	—	1
NLMz	18GH74	—	1	—	—	—
NLMz	18GH78	1	1	1	3	2
NLMz	18GH79	—	2	2	—	1
NLMz	18GH8	1	2	2	1	2
NLMz	18GH9	1	2	1	—	1
TN90	17GH1696	1	1	1	1	1
TN90	17GH1717	1	2	2	2	1
TN90	17GH1719	1	2	1	1	1
TN90	17GH1729	2	1	1	1	1
TN90	17GH1736	1	1	2	1	1
TN90	17GH1737	1	2	2	2	1
TN90	17GH1739	1	1	1	1	2
TN90	17GH1740	1	2	1	2	1
TN90	17GH1835	2	2	2	1	1
TN90	17GH1848	1	2	1	1	1
TN90	17GH1849	1	1	1	2	2
TN90	17GH1912	1	2	1	1	1
TN90	17GH1937	1	2	1	2	1
TN90	17GH1940	1	2	1	1	1
TN90	17GH1943	1	1	1	1	2
TN90	17GH1944	1	1	1	1	2
TN90	18GH1051	2	2	2	1	2
TN90	18GH22	1	2	1	2	1
TN90	18GH34	1	1	1	1	2
TN90	18GH473	1	1	1	2	2
TN90	18GH49	1	1	1	1	1
TN90	18GH50	2	1	1	1	1
TN90	18GH848	2	2	2	1	2
TN90	18GH850	1	2	1	2	2
TN90	18GH851	1	2	1	2	2
TN90	17GH1699	3	2	2	2	1
TN90	17GH1708	1	3	1	2	—
TN90	17GH1722	2	1	2	2	1
TN90	17GH1724	2	1	1	2	1
TN90	17GH1725	2	1	1	2	1
TN90	17GH1845	2	2	1	2	2
TN90	17GH1846	2	1	2	2	1
TN90	17GH1847	2	1	2	2	1
TN90	17GH1911	1	2	1	1	1
TN90	17GH1912	1	2	1	1	1
TN90	17GH1915	—	1	—	1	—
TN90	17GH1918	2	2	1	1	5
TN90	17GH1928	2	2	—	2	1
TN90	17GH1932	2	2	—	—	1
TN90	17GH1933	2	2	2	5	1
TN90	17GH1936	2	2	2	1	2
TN90	18GH20	—	1	2	1	2
TN90	18GH28	2	1	2	1	2
TN90	18GH31	1	3	1	1	1
TN90	18GH47	1	3	1	1	1
TN90	18GH51	—	—	—	1	—
TN90	18GH52	—	—	—	1	—
Number one (1) represents homozygous for a single mutant allele. Numbers 2 to 5 represent a heteroallelic combination having 2 to 5 Indels. Hyphens indicate no data. Detailed genotype information is shown in Tables 4A to 4D.

TABLE 4A
Mutantpmt alleles in K326 produced by genome editing using GET2. The position of
each edited site (e.g.,, indels) is relative to the nucleotide number on the corresponding
cDNA sequence of each PMT gene. For example, line 17GH1678 has bi-allelic mutations in PMT1b.
One of the two alleles has a four-nucleotide deletion which corresponds to nucleotides 416 to
419 of the PMT1b cDNA sequence. The other allele has a two-nucleotide deletion which
corresponds to nucleotides 418 to 419 of the PMT1b cDNA sequence. SEQ ID Numbers are
assigned and shown for sequences of more than 10 nucleotides.

PMT1b

PMT1a

PMT2

PMT3

PMT4

VAR-		Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted
IETY	LINE	ition	sequence	ition	sequence	ition	sequence	ition	sequence	ition	sequence
K326	17GH	416..	ATAC	415..	CATAC	348..	AC	432..	ACAC	547.	CACAC
	1678	419		421	AG	349		435		.551
		418..	AC	417..	TACA					548.	ACAC
		419		420						.551
K326	17GH	414..	ACAT	414..	ACAT	348..	AC	432..	ACAC	546..	AC
	1680	417		417		349		435		547
				416..	AT
				417
K326	17GH	414..	ACAT	411..	TCAAC	348..	AC	433..	CACAC	548..	ACACA
	1804	417		420	ATACA	349		437		552
					(SEQ ID
					NO: 379)
				417..	TACA
				420
K326	17GH	414..	ACAT	411..	TCAAC	348..	AC	433..	CACAC	548..	ACACA
	1898	417		420	ATACA	349		437		552
					(SEQ ID
					NO: 379)
				417..	TACA
				420
K326	18GH	414..	ACAT	415..	C	348..	ACAC	432..	AC	546..	ACAC
	207	417		415		351		433		549
				417..	T
				417
K326	18GH	414..	ACAT	414..	ACAT	348..	AC	432..	ACAC	546..	AC
	343	417		417		349		435		547
				416..	AT
				417
K326	18GH	414..	ACAT	414..	ACAT	348..	AC	429..	TCAACACAC	546..	AC
	348	417		417		349		439	AG	547
									(SEQ ID
									NO: 396)
K326	18GH	414..	ACAT	414..	ACAT	348..	AC	429..	TCAACACAC	546..	AC
	349	417		417		349		439	AG	547
									(SEQ ID
									NO: 396)
K326	18GH	414..	ACAT	414..	ACAT	348..	ACAC	431..	AACACACA	546..	AC
	355	417		417		351		438		547
				416..	AT	350..	AC	435..	CACA	550..	ACAG
				417		351		438		553
								440..	AGA
								442
K326	18GH	414..	ACAT	411..	TCAAC	348..	AC	433..	CACAC	548..	ACACA
	359	417		420	ATACA	349		437		552
					(SEQ ID
					NO: 379)
				417..	TACA
				420
K326	18GH	413..	AACA	414..	ACAT	349..	CACA	432..	AC	546..	ACAC
	64	420	TACA	417		352		433		549
		417..	TACA			354..	AGAG
		420				359	AA
K326	18GH	415..	CATA	413..	AACAT	348..	ACAC	432..	ACACACAG	543..	TCAACA
	682	421	CAG	422	ACAGA	351		439		554	CACAGA
					(SEQ ID						(SEQ ID
					NO: 386)						NO: 404)
				417..	TACA	350..	AC	437..	CA	549..	CACA
				420		351		438		552
K326	18GH	414..	ACAT	414..	ACATA	348..	ACAC	432..	ACACACAG	546..	ACAC
	692			420	CA	351		439		549
		417		416..	ATACA	350..	AC	437..	CA
				420		351		438
K326	18GH	414..	ACAT	414..	ACAT	348..	ACAC	430..	CAACACA	546..	ACAC
	697	417		417		351		436		549
K326	18GH	414..	ACAT	414..	ACAT	348..	ACAC	430..	CAACACA	546..	ACAC
	922	417		417		351		436		549
K326	18GH	414..	ACAT	414..	ACAT	349..	CACA	431..	AACACACA	546..	AC
	957	417		417		355	CAG	438		547
						351..	CACA
						354
K326	17G	—	—	—	—	346.	CAAC	432.	AC	—	—
	6					.350	A
	H188					349.	CA	.433
						.350
K326	17GH	—	—	418..	AC	348..	AC	—	—	—	—
	1888			419		349
K326	17GH	—	—	418..	AC	348..	AC	—	—	—	—
	1889			419		349
K326	17GH	413..	AACA	414..	ACAT	348..	ACAC	432..	ACAC
	1892	419	TAC	417		351		435
		414..	ACAT			350..	AC	434..	AC
		419	AC			351		435
		416..	ATAC
		419
K326	17GH	416..	ATAC	416..	ATACA	348..	AC	430..	CAACACA	546..	AC
	1893	421	AG	420		349		436		547
K326	17GH	414..	ACAT	417..	TACA	348..	ACAC	432..	ACAC	548..	ACACA
	1901	417		420		355		435		552
							ACAG	434..	AC	550..	ACA
								435		552
K326	17GH	414..	ACAT	417..	TACA	348..	ACAC	432..	ACAC	548..	ACACA
	1902	417		420		355		435		552
							ACAG	434..	AC	550..	ACA
								435		552
K326	17GH	414..	ACAT	413..	AACAT	352..	ACA	432..	ACAC	546..	AC
	1808	417		421	ACAG	354		435		547
				418..	ACAG			434..	AC
				421				435
K326	17G	414..	ACAT	417..	TACA	348..	ACAC	432..	ACAC	548..	ACACA
	1H810	417		420		355		435		552
							ACAG	434..	AC	550..	ACA
								435		552
K326	18GH3	—	—	414..	ACAT	348..	AC	432..	ACAC	—	—
				417		349		435
K326	18GH4	—	—	414..	ACAT	348..	AC	429..	TCAACACAC	546..	AC
				417		349		439	AG	547
									(SEQ ID
									NO: 396)

TABLE 4B
Mutantpmt alleles in TN90 produced by genome editing using GET2.

PMT1b

PMT1a

PMT2

PMT3

PMT4

VAR-		Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted
IETY	LINE	ition	sequence	ition	sequence	ition	sequence	ition	sequence	ition	sequence
TN90	17GH	414..	ACAT	414..	ACAT	348..	AC	436..	ACAG	546..	AC
	1696	417		417		349		439		547
TN90	17GH	414..	ACAT	414..	ACAT	346..	CAACACA	432..	ACAC	546..	AC
	1717	417		417		352		435		547
				415..	CA	349..	CACA	434..	AC
				416		352		435
TN90	17GH	414..	ACAT	417..	TACA	348..	AC	432..	AC	546..	ACA
	1719	417		420		349		433		549	C
				417..	TACA
				423	GAG
TN90	17GH	412..	CAAC	412..	CAAC	348..	ACAC	432..	AC	546..	AC
	1729	421	ATACAG	418	ATA	351		433		547
			(SEQ
			ID NO:
			380)
		414..	ACAT
		420	ACA
TN90	17GH	418..	ACAG	414..	ACAT	347..	AACACACA	432..	AC	546..	AC
	1736	421		417		357	GAG (SEQ	433		547
							ID
							NO: 391)
						351..	CACA
						354
TN90	17GH	414..	ACAT	414..	ACAT	346..	CAACACA	432..	ACAC	546..	AC
	1737	417		417		352		435		547
				415..	CA	349..	CACA	434..	AC
				416		352		435
TN90	17GH	414..	ACAT	414..	ACAT	348..	AC	432..	AC	546..	ACA
	1739	417		417		349		433		549	C
										548..	AC
										549
TN90	17GH	414	ACAT	416..	ATAC	348..	ACAC	435..	CACA	546..	AC
	1740	417..		419		351		438		547
				418..	AC			436..	ACAG
				419				439
TN90	17GH	413..	AACA	417..	TACA	350..	ACACA	432..	ACAC	546..	AC
	1835	421	TACA	420		354		435		547
			G
		417..	TACA	418..	ACAG	351..	CACAGAGA
		420		421		362	ATGG
							(SEQ ID
							NO: 394)
TN90	17GH	414..	ACAT	417..	TACA	348..	AC	432..	AC	546..	ACA
	1848	417		420		349		433		549	C
				417..	TACA
				423	GAG
TN90	17G	414	ACAT	414	ACAT	348	ACAC	430	CAACACA	546	ACA
	49	7		7		1		..43		..54	C
								6		9
	H18	..41		..41		..35		433	CACA	548	AC
								..43		..54
								6		9
TN90	17GH	414..	ACAT	417..	TACA	348..	AC	432..	AC	546..	ACA
	1912	417		420		349		433		549	C
				417..	TACA
				423	GAG
TN90	17GH	416..	ATAC	412..	CAAC	348..	ACAC	432..	ACAC	546..	AC
	1937	419		421	ATACAG	351		435		547
					(SEQ
					ID NO:
					380)
				417..	TACA			434..	AC
				420				435
TN90	17GH	414..	ACAT	417..	TACA	348..	AC	432..	AC	546..	ACA
	1940	417		420		349		433		549	C
				417..	TACA
				423	GAG
TN90	17GH	414..	ACAT	414..	ACAT	348..	AC	432..	AC	546..	ACA
	1943	417		417		349		433		549	C
										548..	AC
										549
TN90	17GH	414..	ACAT	414..	ACAT	348..	AC	432..	AC	546..	ACA
	1944	417		417		349		433		549	C
										548..	AC
										549
TN90	18GH	414..	ACAT	412..	CAAC	348..	ACAC	432..	AC	546..	ACA
	1051	418	A	418	ATA	351		433		549	C
		415..	CATA	415..	CATA	350..	AC			548..	AC
		421	CAG	418		351				549
TN90	18GH	416..	ATAC	412..	CAAC	348..	ACAC	432..	ACAC	546..	AC
	22	419		421	ATACAG	351		435		547
					(SEQ
					ID NO:
					380)
				417..	TACA			434..	AC
				420				435
TN90	18GH	414..	ACAT	414..	ACAT	348..	AC	432..	AC	546..	ACA
	34	417		417		349		433		549	C
										548..	AC
										549
TN90	18GH	414..	ACAT	414..	ACAT	348..	ACAC	430..	CAACACA	546..	ACA
	473	417		417		351		436		549	C
								433..	CACA	548..	AC
								436		549
TN90	18GH	412..	CAAC	412..	CAAC	348..	ACAC	432..	AC	546..	AC
	49	421	ATACAG	418	ATA	351		433		547
			(SEQ
			ID NO:
			380)
TN90	18GH	412..	CAAC	412..	CAAC	348	ACAC	432..	AC	546..	AC
	50	421	ATACAG	418	ATA	351..		433		547
			(SEQ
			ID NO:
			380)
		414..	ACAT
		420	ACA
TN90	18GH	414..	ACAT	412..	CAAC	348..	ACAC	432..	AC	546..	ACA
	848	418	A	418	ATA	351		433		549	C
		415..	CATA	415..	CATA	350..	AC			548..	AC
		421	CAG	418		351				549
TN90	18GH	414..	ACAT	416..	ATAC	348..	AC	435..	CACA	546..	AC
	850	417		422	AGA	349		438		547
				417..	TACA			436..	ACAG	550..	ACA
				420				439		553	G
TN90	18GH	414..	ACAT	416..	ATAC	348..	AC	435..	CACA	546..	AC
	851	417		422	AGA	349		438		547
				417..	TACA			436..	ACAG	550..	ACA
				420				439		553	G
TN90	17GH	419..	CA	413..	AACA	348..	ACAC	429..	TCAACACA	546..	AC
	1699	420		420	TACA	351		438	CA	547
									(SEQ ID
									NO: 395)
		418..	ACAG	419..	CA	349..	C	432..	ACACACAG
		423	AG	420		349		446	AGAAT
									GG (SEQ
		427..	G						ID NO:
		427							399)
TN90	17GH	414..	ACAT	414..	AC	346..	CAACACA	432..	ACACAC	-	-
	1708	417		415		355	CAG	437
				418..	ACAG		(SEQ ID	440..	AGAA
				424	AGA		NO: 390)	443
				419..	CA
				420
TN90	17GH	415..	CATA	414..	ACAT	348..	ACAC	432..	AC	546..	ACA
	1722	420	CA	417		351		433		549	C
		418..	ACAG			350..	AC	435..	CACAG
		421				351		439
TN90	17GH	416..	ATAC	414..	ACAT	348..	ACAC	432..	ACAC	550..	ACA
	1724	421	AG	417		351		435		553	G
		417..	TACA					434..	AC
		420						435
TN90	17GH	416..	ATAC	414..	ACAT	348..	ACAC	432..	ACAC	550..	ACA
	1725	421	AG	417		351		435		553	G
		417..	TACA					434..	AC
		420						435
TN90	17GH	416..	ATA	412..	CAAC	348..	ACAC	433..	CACAC	546..	ACA
	1845	418		418	ATA	351		437		551	CAC
		418..	AC	415..	CATA			436..	AC	550..	AC
		419		418				437		551
TN90	17GH	415..	CATA	414..	ACAT	348..	ACAC	432..	AC	546..	ACA
	1846	420	CA	417		351		433		549
		418..	ACAG			350..	AC	435..	CACAG		C
		421				351		439
TN90	17GH	415..	CATA	414..	ACAT	348..	ACAC	432..	AC	546..	ACA
	1847	420	CA	417		351		433		549	C
		418..	ACAG			350..	AC	435..	CACAG
		421				351		439
TN90	17GH	414..	ACAT	417..	TACA	348..	AC	432..	AC	546..	ACA
	1911	417		420		349		433		549	C
				417..	TACA
				423	GAG
TN90	17GH	414..	ACAT	417..	TACA	348..	AC	432..	AC	546..	ACA
	1912	417		420		349		433		549	C
				417..	TACA
				423	GAG
TN90	17GH	—	—	414..	ACAT	—	—	432..	ACAC	—	—
	1915			417				435
TN90	17GH	414..	ACAT	417..	TACA	353..	CAGAGAAT	432..	ACAC	544..	CAA
	1918	419	AC	420		361	G	435		550	CACA
										554..	A
										554
		416..	ATAC	418..	ACAG					558..	TGG
		419		421						563	TGG
										565..	TT
										566
										569..	CAT
										572	A
TN90	17GH	412..	CAAC	414..	ACAT			432..	ACACACAG	546..	AC
	1928	418	ATA	417				448	AGAATGGT	547
									G
									(SEQ ID
									NO: 400)
		415..	CATA	419..	CAG			437..	CA
		418		421				438
TN90	17GH	414..	ACAT	416..	ATAC	—	—	—	—	546..	ACA
	1932	419	AC	419						551	CAC
		416..	ATAC	418..	AC
		419		419
TN90	17GH	414..	ACAT	416..	ATAC	350..	ACACAG	413..	CT	546..	ACA
	1933	419	AC	419		355		414		551	CAC
								418..	GA
								419
		416..	ATAC	418..	AC	351..	CACA	426..	AA
		419		419		354		427
								431..	AA
								432
								432..	ACAC
								435
TN90	17GH	413..	AACA	416..	ATAC	348..	ACAC	432..	AC	544..	CAA
	1936	421	TACA	419		351		433		550	CAC
			G								A
		417..	TACA	418..	AC	350..	AC			547..	CAC
		420		419		351				550	A
TN90	18GH			414..	ACAT	348..	AC	432..	AC	546..	ACA
	20			419	AC	349		433		549	C
						352..	ACAG			548..	AC
						355				549
TN90	18GH	414..	ACAT	416..	ATAC	348..	AC	436..	ACAG	546..	ACA
	47	419	AC	419		349		439		549	C
				418..	AC
				419
				424..	AA
				425
TN90	18GH	414..	ACAT	414..	ACAT	348..	ACAC	433..	CACAC	546..	ACA
	28	419	AC	417		351		437		549	C
		416..	ATAC			350..	AC			548..	AC
		419				351				549
TN90	18GH	414..	ACAT	416..	ATAC	348..	AC	436..	ACAG	546..	ACA
	31	419	AC	419		349		439		549	C
				418..	AC
				419
				424..	AA
				425

TABLE 4C
Mutant pmt alleles in NLMz produced by genome editing using GET2. NLMz refers
to the Narrow Leaf Madole variety containing triple loss-of-function mutations in three
nicotine demethylase genes (CYP82E4, CYP82E5v2, and CYP82E10).

PMT1b

PMT1a

PMT2

PMT3

PMT4

VA-		Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted
RIETY	LINE	ition	sequence	ition	sequence	ition	sequence	ition	sequence	ition	sequence
NL	18GH10	414..	ACAT	414..	ACAT	350..	AC	431..	AACACACA	546..	ACAC
Mz		417		417		351		441	GAG (SEQ	549
									ID NO:
									391)
NL	18GH	414..	ACAT	412..	CAACATA	348..	AC	430..	CAACACA	546..	ACAC
Mz	1004	417		418		349		436		553	ACAG
		416..	A					435..	CA	551..	CA
		416						436		552
NL	18GH	414..	ACAT	412..	CAACATA	348..	AC	430..	CAACACA	546..	ACAC
Mz	1033	417		418		349		436		553	ACAG
		416..	A					435..	CA	551..	CA
		416						436		552
NL	18GH	417..	TA	416..	ATAC	348..	ACACA	432..	ACACAC	546..	AC
Mz	132	418		419		352		437		547
				418..	AC	348..	ACACAC	434..	ACAC
				419		353		437
								436..	AC
								437
NL	18GH	414..	ACAT	414..	ACATACAG	348..	ACAC	433..	CACACAG	550..	ACAG
Mz	134	417		423	AG (SEQ	351		439		556	AGA
					ID NO:
					388)
				419..	CA
				420
NL	18GH	414..	ACAT	415..	CATAC	346..	CAACACA	432..	ACAC	545..	AACA
Mz	217	417		419		352		435		557	CACA
											GAGA
											A
											(SEQ
											ID NO:
											407)
				417..	TA	351..	CA			551..	CA
				418		352				552
NL	18GH	416..	ATAC	414..	ACAT	348..	AC	432..	AC	546..	ACAC
Mz	456	419		417		349		433		549
		418..	AC
		419
NL	18GH	414..	ACAT	414..	ACAT	348..	AC	432..	AC	546..	ACAC
Mz	457	417		417		349		433		549
NL	18GH	414..	ACAT	409..	ATTCAACA	350..	ACACAGAG	436..	ACAG	550..	ACAG
Mz	460	417		420	TACA (SEQ	363	AATGGT	439		553
					ID NO:		(SEQ ID
					383)		NO: 393)
				416..	ATACAGAG	351..	CACA
				429	AATGGT	354
					(SEQ ID	353..	CA
					NO: 389)	354
NL	18GH	414..	ACAT	412..	CAACATA	348..	AC	430..	CAACACA	546..	ACAC
Mz	465	417		418		349		436		553	ACAG
		416..	A					435..	CA	551..	CA
		416						436		552
NL	18GH	414..	ACAT	414..	ACAT	348..	ACAC	432..	ACAC	550..	ACAG
Mz	830	417		417		351		435		553
NL	18GH	413..	AACATACA	413..	AACATACA	348..	ACAC			546..	AC
Mz	831	428	GAGAATGG	428	GAGAATGG	351				547
			(SEQ ID		(SEQ ID
			NO: 381)		NO: 381)
				417..	TACA
				420
NL	18GH	414..	ACAT	414..	ACAT	348..	AC	432..	AC	546..	ACAC
Mz	836	417		417		349		433		549
NL	18GH	415..	CATACAG	413..	AACATACA	347..	AACACACA			546..	AC
Mz	841	421		420		357	GAG (SEQ			547
							ID NO:
							391)
		419..	CA	414..	ACATACA
		420		420
NL	18GH	411..	TCAACATA	411..	TCAACATA	351..	CACA	432..	AC	546..	ACAC
Mz	974	420	CA (SEQ	420	CA (SEQ	354		433		549
			ID NO:		ID NO:
			379)		379)
		417..	TACA			353..	CA	436..	ACAG
		420				354		439
NL	18GH	412..	CAACATA	414..	ACAT	346..	CAACACA	429..	TCAACACA	546..	ACAC
Mz	981	418		417		352		439	CAG (SEQ	552	ACA
									ID NO:
									396)
						349..	CACA	431..	AACACACA	546..	ACAC
						352		441	GAG (SEQ	553	ACAG
									ID NO:
									391)
NL	18GH	414..	ACAT	412..	CAACATA	348..	AC	430..	CAACACA	546..	ACAC
Mz	994	417		418		349		436		553	ACAG
		416..	A					435..	CA	551..	CA
		416						436		552
NL	18GH			414..	ACATACA	348..	ACAC			546..	AC
Mz	128			420		351				547
				417..	TACAG	350..	AC
				421		351
NL	18GH	414..	ACATACAG	412..	CAACATA	349..	CACACAG	430..	CAACACA	546..	ACAC
Mz	130	437	AGAATGGT	418		355		436		549
			GGATTTCC
			(SEQ ID
			NO: 382)
		417..	TACA	415..	CATA
		420		418
NL	18GH	417..	TA	416..	ATAC	348..	ACACAC	—	—	546..	AC
Mz	131	418		419		353				547
				417..	TAC	350..	ACAC
				419		353
				418..	AC
				419
NL	18GH	413..	AACATAC	414..	ACATACA	348..	ACAC	—	—	546..	AC
Mz	133	419		420		351				547
		414..	ACATAC	417..	TACA	350..	AC
		419		420		351
				417..	TACAG
				421
NL	18GH	—	—	—	—	348..	AC	—	—	—	—
Mz	136					349
NL	18GH	412..	CAACATA	414..	ACATAC	347..	AACACACA	432..	AC	546..	ACAC
Mz	216	418		419		354		433		549
		415..	CATA	416..	ATAC					548..	AC
		418		419						549
NL	18GH	418..	AC	414..	ACAT	348..	ACAC			546..	ACAC
Mz	227	419		417		351				549
NL	18GH	414..	ACAT	414..	ACATACA	352..	ACAG	429..	TCAACAC	546..	ACAC
Mz	5	417		420		355		435		551	AC
				415..	CATACAG			432..	ACAC	548..	ACAC
				421				435		551
NL	18GH	414..	ACAT	416..	ATACAGAG	350..	ACACAGAG	436..	ACAG	550..	ACAG
Mz	6	417		429	AATGGT	363	AATGGT	439		553
					(SEQ ID		(SEQ ID
					NO: 389)		NO: 393)
				417..	TACAGA	351..	CACAGA
				422		356
						353..	CAGA
						356
NL	18GH	416..	ATACAGAG	414..	ACATAC	348..	ACAC	433..	CACAC	546..	ACAC
Mz	65	423		419		351		437		549
		418..	ACA	417..	TAC	350..	AC	436..	AC
		420		419		351		437
NL	18GH	414..	ACAT	414..	ACATACA	352..	ACAG	429..	TCAACAC	546..	ACAC
Mz	66	417		420		355		435		551	AC
				415..	CATACAG			432..	ACAC	548..	ACAC
				421				435		551
NL	18GH			411..	TCAACATA			432..	AC	546..	ACAC
Mz	69			420	CA (SEQ			433		549
					ID NO:			436..	ACAG
					379)			439
NL	18GH	416..	ATACAGAG	414..	ACATAC	348..	ACAC	433..	CACAC	546..	ACAC
Mz	72	423		419		351		437		549
		418..	ACA	417..	TAC	350..	AC	436..	AC
		420		419		351		437
NL	18GH	—	—	414..	ACATACA	348..	ACAC	—	—	546..	AC
Mz	73			420		351				547
				417..	TACAG	350..	AC
				421		351
NL	18GH	—	—	412..	CAACATA	—	—	—	—	—	—
Mz	74			418
NL	18GH	414..	ACATAC	414..	ACAT	348..	AC	431..	A	546..	ACAC
Mz	78	419		417		349		431		549
								434..	ACACA	548..	AC
								438		549
								435..	CACA
								438
NL	18GH	—	—	414..	ACATACA	348..	ACAC	—	—	546..	AC
Mz	79			420		351				547
				417..	TACAG	350..	AC
				421		351
NL	18GH	417..	TACA	416..	ATACAG	348..	ACACACA	435..	CACAGAG	549..	CACA
Mz	8	420		421		354		447	AATGGT	552
									(SEQ ID
									NO: 401)
				417..	TACA	350..	ACACA			549..	CACA
				420		354				553	G
NL	18GH	417..	TA	416..	ATAC	348..	ACACAC	—	—	546..	AC
Mz	9	418		419		353				547
				418..	AC
				419
NL	18GH	414..	ACAT	414..	ACAT	348..	ACAC	431..	AACACACA	546..	ACAC
Mz	71	417		417		351		441	GAG (SEQ	549
									ID NO:
									391)
NL	17GH	414..	ACAT	414..	ACATACA	352..	ACAG	429..	TCAACAC	546..	ACAC
Mz	1905	417		420		355		435		551	AC
				415..	CATACAG			432..	ACAC	548..	ACAC
				421				435		551

TABLE 4D
Mutant pmt alleles in oriental tobacco produced by genome editing using GET2.

PMT1b

PMT1a

PMT2

PMT3

PMT4

		Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted	Pos-	Deleted
VARIETY	LINE	ition	sequence	ition	sequence	ition	sequence	ition	sequence	ition	sequence
Kat-	18GH	412..	CAAC	416..	ATAC	348..	ACACA	432..	ACAC	546..	AC
erini	125	418	ATA	419		355	CAG	435		547
		414..	ACAT	418..	AC			434..	AC
		418	A	419				435
Basma	18GH	414..	ACAT	412..	CAAC	348..	ACACA	432..	ACAC	546..	ACAC
	203	417		418	ATA	357	CAGAG	435		549
							(SEQ ID
							NO: 392)
				415..	CATA	353..	CA	434..	AC
				418		354		435
Kat-	18GH	416..	ATAC	414..	ACAT	352..	ACAG	432..	ACACAC	546..	ACACACAG
erini	208	419		417		355		441	AGAG	553
									(SEQ ID
									NO: 392)
		418..	AC					435..	CACA
		419						438
Basma	18GH	414..	ACAT	412..	CAAC	348..	ACACA	432..	ACAC	546..	ACAC
	134	417		418	ATA	357	CAGAG	435		549
							(SEQ ID
							NO: 392)
				415..	CATA	353..	CA	434..	AC
				418		354		435
Kat-	18GH	414..	ACAT	414..	ACAT	348..	ACAC	432..	ACAC
erini	403	417		417		351		435
Kat-	18GH	412..	CAAC	414..	ACAT	348..	AC	430..	CAACAC	546..	AC
erini	414	418	ATA	417		349		436	A	547
		415..	CATA
		418
Kat-	18GH	413..	AACA	414..	ACAT	351..	CACAG	433..	CACACA	546..	AC
erini	434	420	TACA	417		357	AG	439	G	547
		417..	TACA					437..	CA
		420						438
Kat-	18GH	412..	CAAC	414..	ACAT	346..	CAACA	432..	AC	546..	AC
erini	436	418	ATA	421	ACAG	352	CA	433		547
		415..	CA					436..	ACAGAG
		416						443	AA
								445..	GGTGG
								449
								451..	TTTCCAT
								463	ACACTG
									(SEQ ID
									NO: 402)
Kat-	18GH	414..	ACAT	414..	ACAT	348.	AC	433..	CACACA	544..	CAACACAC
erini	437	417		417				439	G	553	AG (SEQ
											ID NO:
											390)
				416..	AT	.349		435..	CACA	551..	CA
				417				438		552
Kat-	H18G	414..	ACAT	413..	AACA	348..	AC	432..	ACAC	546..	ACAC
erini	449	417		419	TAC	349		435		549
		415..	CA	418..	AC
		416		419
Kat-	18GH	416..	ATAC	414..	ACAT	348..	ACAC	432..	ACACAC	545..	AACACACA
erini	706	420	A	417		351		439	AG	555	GAG
		419..	CA					435..	CACA		(SEQ ID
		420						438			NO: 391)
Kat-	18GH	412..	CAAC	414..	ACAT	348..	AC	432..	ACAC	546..	AC
erini	709	418	ATA	417		349		435		547
		417..	TA	416..	AT
		418		417
Kat-	18GH	414..	ACAT	414..	ACAT	351..	CACA	432..	ACAC	546..	AC
erini	710	417		417		354		435		547
						352..	ACAG	434..	AC
						355		435
Kat-	18GH	416..	ATAC	417..	TACA	348..	ACAC	432..	ACACAC	546..	ACAC
erini	716	419		420		351		439	AG	549
		418..	AC	417..	TACA			435..	CACA	548..	AC
		419		421	G			438		549
Kat-	18GH	414..	ACAT	414..	ACAT	350..	ACACA	432..	ACAC	546..	AC
erini	729	417		417		357	GAG	435		547
						353..	CA
						354
Kat-	18GH	414..	ACAT	414..	ACAT	348..	ACAC	433..	C	544..	CAACACAC
erini	731	417		417		351		433		553	AG (SEQ
								435..	CACAG		ID NO:
								439			390)
Kat-	18GH	416..	ATAC	414..	ACAT	348..	AC	432..	ACAC	546..	ACAC
erini	752	419		417		349		435		549
		418..	AC
		419
Kat-	18GH	416..	ATAC	414..	ACAT	353..	CA	433..	CACACA	545..	AACACACA
erini	756	419		417		354		439	G	555	GAG (SEQ
											ID NO:
											391)
		416..	ATAC	414..	ACAT	353..	CA	433..	CACACA	545..	AACACACA
		419		417		354		439	G	555	GAG
											(SEQ ID
											NO: 391)
								437..	CA	549..	CACA
								438		552
Kat-	18GH	416..	ATAC	414..	ACAT	348..	ACAC	432..	AC	544..	CAACACA
erini	768	419		417		351		433		550
										547..	CACA
										550
Kat-	18GH	416..	ATAC	414..	ACAT	346..	CAACA	432..	ACAC	546..	AC
erini	771	419		417		352	CA	435		547
						349..	CACA	434..	AC
						352		435
Kat-	18GH	409..	ATTC	411..	TCAA	348..	ACAC	441..	G	546..	ACAC
erini	776	415	AAC	417	CAT	351		441		549
		418..	AC	414..	ACAT					548..	AC
		419		417						549
Kat-	18GH	412..	CAAC	414..	ACAT	348..	ACAC	432..	ACAC	541..	ATTCAACA
erini	800	418	ATA	420	ACA	351		435		551	CAC (SEQ
											ID NO:
											403)
		415..	CATA	416..	ATAC					548..	ACAC
		418		420	A					551
Kat-	18GH	414..	ACAT	409..	ATTC	348..	ACAC	432..	ACAC	—	—
erini	818	417		415	AAC	351		435
								434..	AC
								435

TABLE 4E
Mutant pmt alleles in NLM (Ph Ph) tobacco produced by genome editing using GET1.

PMT1b

PMT1a

PMT2

PMT3

PMT4

		Position		Position		Position		Position		Position
		from		from		from		from		from
LINE	Modification	ATG		ATG		ATG		ATG		ATG

CS15	A-deleted	131	A-inserted	132	A-deleted	98	A-deleted	98	A-del	131
	T-deleted	262			T-deleted	196	T-deleted	282

TABLE 5A
A list of exemplary mutant alleles obtained in the PMTIb gene. Mutant allele
sequences listed here and Tables 5B to 5E represent about 40-nucleotide-long genomic
sequences from each edited PMT gene with the edited site in the middle of the genomic sequence
(e.g., 20 nucleotides on each side of the deleted or inserted sequence site). These mutant alleles
corresponds to those listed in Tables 4A to 4E.
PMT1b

		Mutant		Reference
	Deleted	Allele		Allele
	sequence	Sequence		Sequence
Pos-	(SEQ ID	(SEQ ID	Mutant	(SEQ ID
ition	No.)	No.)	Allele Sequence	No.)	Reference Allele Sequence
131...	A	23	TGGCATTTCCAAACACCAA	201	TGGCATTTCCAAACACCAAAaCGGGCACCA
131			ACGGGCACCAGAATGGCAC		GAATGGCACTT
			TT
262...	T	24	CCAACTCTATTAAGCCTGGT	202	CCAACTCTATTAAGCCTGGTtGGTTTTCAGA
262			GGTTTTCAGAGTTTAGCGCA		GTTTAGCGCA
409..	ATTCAAC	25	TTCTGACTTTGGATGGAGCA	203	TTCTGACTTTGGATGGAGCAattcaacATACAG
415			ATACAGAGAATGGTGGATT		AGAATGGTGGATTT
			T
411..	TCAACATACA	26	CTGACTTTGGATGGAGCAA	204	CTGACTTTGGATGGAGCAATtcaacatacaGAGA
420	(379)		TGAGAATGGTGGATTTCCA		ATGGTGGATTTCCATA
			TA
412..	CAACATA	27	TGACTTTGGATGGAGCAAT	205	TGACTTTGGATGGAGCAATTcaacataCAGAGA
418			TCAGAGAATGGTGGATTTC		ATGGTGGATTTCCA
			CA
412..	CAACATACAG	28	TGACTTTGGATGGAGCAAT	206	TGACTTTGGATGGAGCAATTcaacatacagAGAA
421	(380)		TAGAATGGTGGATTTCCAT		TGGTGGATTTCCATAC
			AC
413..	AACATAC	29	GACTTTGGATGGAGCAATT	207	GACTTTGGATGGAGCAATTCaacatacAGAGAA
419			CAGAGAATGGTGGATTTCC		TGGTGGATTTCCAT
			AT
413..	AACATACA	30	GACTTTGGATGGAGCAATT	208	GACTTTGGATGGAGCAATTCaacatacaGAGAA
420			CGAGAATGGTGGATTTCCA		TGGTGGATTTCCATA
			TA
413..	AACATACAG	31	GACTTTGGATGGAGCAATT	209	GACTTTGGATGGAGCAATTCaacatacagAGAA
421			CAGAATGGTGGATTTCCAT		TGGTGGATTTCCATAC
			AC
413..	AACATACAGAGA	32	GACTTTGGATGGAGCAATT	210	GACTTTGGATGGAGCAATTCaacatacagagaa
428	ATGG(381)		CTGGATTTCCATACACTGAA		tggTGGATTTCCATACACTGAAA
			A
414..	ACAT	33	ACTTTGGATGGAGCAATTC	211	ACTTTGGATGGAGCAATTCAacatACAGAGA
417			AtACAGAGAATGGTGGATTT		ATGGTGGATTTCC
			CC
414..	ACATA	34	ACTTTGGATGGAGCAATTC	212	ACTTTGGATGGAGCAATTCAacataCAGAGAA
418			ACAGAGAATGGTGGATTTC		TGGTGGATTTCCA
			CA
414..	ACATAC	35	ACTTTGGATGGAGCAATTC	213	ACTTTGGATGGAGCAATTCAacatacAGAGAA
419			AAGAGAATGGTGGATTTCC		TGGTGGATTTCCAT
			AT
414..	ACATACA	36	ACTTTGGATGGAGCAATTC	214	ACTTTGGATGGAGCAATTCAacatacaGAGAAT
420			AGAGAATGGTGGATTTCCA		GGTGGATTTCCATA
			TA
414..	ACATACAGAGAA	37	ACTTTGGATGGAGCAATTC	215	ACTTTGGATGGAGCAATTCAacatacagagaat
437	TGGTGGATTTCC		AATACACTGAAATGATTGT		ggtggatttccATACACTGAAATGATTGTTC
	(382)		TC
415..	CA	38	CTTTGGATGGAGCAATTCA	216	CTTTGGATGGAGCAATTCAAcaTACAGAGAA
416			ATACAGAGAATGGTGGATT		TGGTGGATTTC
			TC
415..	CATA	39	CTTTGGATGGAGCAATTCA	217	CTTTGGATGGAGCAATTCAAcataCAGAGAAT
418			ACAGAGAATGGTGGATTTC		GGTGGATTTCCA
			CA
415..	CATACA	40	CTTTGGATGGAGCAATTCA	218	CTTTGGATGGAGCAATTCAAcatacaGAGAAT
420			AGAGAATGGTGGATTTCCA		GGTGGATTTCCATA
			TA
415..	CATACAG	41	CTTTGGATGGAGCAATTCA	219	CTTTGGATGGAGCAATTCAAcatacagAGAATG
421			AAGAATGGTGGATTTCCAT		GTGGATTTCCATAC
			AC
416..	A	42	TTTGGATGGAGCAATTCAA	220	TTTGGATGGAGCAATTCAACaTACAGAGAA
416			CTACAGAGAATGGTGGATT		TGGTGGATTTC
			TC
416..	ATA	43	TTTGGATGGAGCAATTCAA	221	TTTGGATGGAGCAATTCAACataCAGAGAAT
418			CCAGAGAATGGTGGATTTC		GGTGGATTTCCA
			CA
416..	ATAC	44	TTTGGATGGAGCAATTCAA	222	TTTGGATGGAGCAATTCAACatacAGAGAATG
419			CAGAGAATGGTGGATTTCC		GTGGATTTCCAT
			AT
416..	ATACA	45	TTTGGATGGAGCAATTCAA	223	TTTGGATGGAGCAATTCAACatacaGAGAATG
420			CGAGAATGGTGGATTTCCA		GTGGATTTCCATA
			TA
416..	ATACAG	46	TTTGGATGGAGCAATTCAA	224	TTTGGATGGAGCAATTCAACatacagAGAATG
421			CAGAATGGTGGATTTCCAT		GTGGATTTCCATAC
			AC
416..	ATACAGAG	47	TTTGGATGGAGCAATTCAA	225	TTTGGATGGAGCAATTCAACatacagagAATGG
423			CAATGGTGGATTTCCATAC		TGGATTTCCATACAC
			AC
417..	TA	48	TTGGATGGAGCAATTCAAC	226	TTGGATGGAGCAATTCAACAtaCAGAGAATG
418			ACAGAGAATGGTGGATTTC		GTGGATTTCCA
			CA
417..	TACA	49	TTGGATGGAGCAATTCAAC	227	TTGGATGGAGCAATTCAACAtacaGAGAATG
420			AGAGAATGGTGGATTTCCA		GTGGATTTCCATA
			TA
418..	AC	50	TGGATGGAGCAATTCAACA	228	TGGATGGAGCAATTCAACATacAGAGAATG
419			TAGAGAATGGTGGATTTCC		GTGGATTTCCAT
			AT
418..	ACA	51	TGGATGGAGCAATTCAACA	229	TGGATGGAGCAATTCAACATacaGAGAATGG
420			TGAGAATGGTGGATTTCCA		TGGATTTCCATA
			TA
418..	ACAG	52	TGGATGGAGCAATTCAACA	230	TGGATGGAGCAATTCAACATacagAGAATGG
421			TAGAATGGTGGATTTCCAT		TGGATTTCCATAC
			AC
418..	ACAGAG	53	TGGATGGAGCAATTCAACA	231	TGGATGGAGCAATTCAACATacagagAATGGT
423			TAATGGTGGATTTCCATACA		GGATTTCCATACAC
			c
419..	CA	54	GGATGGAGCAATTCAACAT	232	GGATGGAGCAATTCAACATAcaGAGAATGG
420			AGAGAATGGTGGATTTCCA		TGGATTTCCATA
			TA
427..	G	55	CAATTCAACATACAGAGAA	233	CAATTCAACATACAGAGAATgGTGGATTTC
427			TGTGGATTTCCATACACTGA		CATACACTGAA
			A

TABLE 5B
A list of exemplary mutant alleles obtained in the PMTla gene.
PMT1a

		Mutant		Reference
		Allele		Allele
	Deleted	Sequence		Sequence
Pos-	sequence	(SEQ ID	Mutant	(SEQ ID
ition	(SEQ ID No.)	No.)	Allele Sequence	No.)	Reference Allele Sequence
132...	A inserted	56	GCACTTCCAAACACCAAAACa	234	GCACTTCCAAACACCAAAACGGGCA
132			GGGCACCAGAATGGCACTTT		CCAGAATGGCACTTT
409..	ATTCAAC	57	TTCTGACTTTGGATGGAGCAA	235	TTCTGACTTTGGATGGAGCAattcaacAT
415			TACAGAGAATGGTGGATTT		ACAGAGAATGGTGGATTT
409..	ATTCAACATACA	58	TTCTGACTTTGGATGGAGCAG	236	TTCTGACTTTGGATGGAGCAattcaacatac
420	(383)		AGAATGGTGGATTTCCATA		aGAGAATGGTGGATTTCCATA
411..	TCAACAT	59	CTGACTTTGGATGGAGCAATA	237	CTGACTTTGGATGGAGCAATtcaacatAC
417			CAGAGAATGGTGGATTTCC		AGAGAATGGTGGATTTCC
411..	TCAACATACA	60	CTGACTTTGGATGGAGCAATG	238	CTGACTTTGGATGGAGCAATtcaacataca
420	(384)		AGAATGGTGGATTTCCATA		GAGAATGGTGGATTTCCATA
412..	CAACATA	61	TGACTTTGGATGGAGCAATTC	239	TGACTTTGGATGGAGCAATTcaacataCA
418			AGAGAATGGTGGATTTCCA		GAGAATGGTGGATTTCCA
412..	CAACATACAG	62	TGACTTTGGATGGAGCAATTA	240	TGACTTTGGATGGAGCAATTcaacatacag
421	(385)		GAATGGTGGATTTCCATAC		AGAATGGTGGATTTCCATAC
413..	AACATAC	63	GACTTTGGATGGAGCAATTCA	241	GACTTTGGATGGAGCAATTCaacatacA
419			GAGAATGGTGGATTTCCAT		GAGAATGGTGGATTTCCAT
413..	AACATACA	64	GACTTTGGATGGAGCAATTCG	242	GACTTTGGATGGAGCAATTCaacatacaG
420			AGAATGGTGGATTTCCATA		AGAATGGTGGATTTCCATA
413..	AACATACAG	65	GACTTTGGATGGAGCAATTCA	243	GACTTTGGATGGAGCAATTCaacatacag
421			GAATGGTGGATTTCCATAC		AGAATGGTGGATTTCCATAC
413..	AACATACAGA	66	GACTTTGGATGGAGCAATTCG	244	GACTTTGGATGGAGCAATTCaacatacag
422	(386)		AATGGTGGATTTCCATACA		aGAATGGTGGATTTCCATACA
413..	AACATACAGAG	67	GACTTTGGATGGAGCAATTCT	245	GACTTTGGATGGAGCAATTCaacatacag
428	AATGG (387)		GGATTTCCATACACTGAAA		agaatggTGGATTTCCATACACTGAAA
414..	AC	68	ACTTTGGATGGAGCAATTCAA	246	ACTTTGGATGGAGCAATTCAacATAC
415			TACAGAGAATGGTGGATTT		AGAGAATGGTGGATTT
414..	ACAT	69	ACTTTGGATGGAGCAATTCAA	247	ACTTTGGATGGAGCAATTCAacatACA
417			CAGAGAATGGTGGATTTCC		GAGAATGGTGGATTTCC
414..	ACATAC	70	ACTTTGGATGGAGCAATTCAA	248	ACTTTGGATGGAGCAATTCAacatacAG
419			GAGAATGGTGGATTTCCAT		AGAATGGTGGATTTCCAT
414..	ACATACA	71	ACTTTGGATGGAGCAATTCAG	249	ACTTTGGATGGAGCAATTCAacatacaG
420			AGAATGGTGGATTTCCATA		AGAATGGTGGATTTCCATA
414..	ACATACAG	72	ACTTTGGATGGAGCAATTCAA	250	ACTTTGGATGGAGCAATTCAacatacagA
421			GAATGGTGGATTTCCATAC		GAATGGTGGATTTCCATAC
414..	ACATACAGAG	73	ACTTTGGATGGAGCAATTCAA	251	ACTTTGGATGGAGCAATTCAacatacaga
423	(388)		ATGGTGGATTTCCATACAC		gAATGGTGGATTTCCATACAC
415..	C	74	CTTTGGATGGAGCAATTCAAA	252	CTTTGGATGGAGCAATTCAAcATACA
415			TACAGAGAATGGTGGATTT		GAGAATGGTGGATTT
415..	CA	75	CTTTGGATGGAGCAATTCAAT	253	CTTTGGATGGAGCAATTCAAcaTACA
416			ACAGAGAATGGTGGATTTC		GAGAATGGTGGATTTC
415..	CATA	76	CTTTGGATGGAGCAATTCAAC	254	CTTTGGATGGAGCAATTCAAcataCAG
418			AGAGAATGGTGGATTTCCA		AGAATGGTGGATTTCCA
415..	CATAC	77	CTTTGGATGGAGCAATTCAAA	255	CTTTGGATGGAGCAATTCAAcatacAGA
419			GAGAATGGTGGATTTCCAT		GAATGGTGGATTTCCAT
415..	CATACAG	78	CTTTGGATGGAGCAATTCAAA	256	CTTTGGATGGAGCAATTCAAcatacagA
421			GAATGGTGGATTTCCATAC		GAATGGTGGATTTCCATAC
416..	AT	79	TTTGGATGGAGCAATTCAACtA	257	TTTGGATGGAGCAATTCAACatACAGA
417			CAGAGAATGGTGGATTTCC		GAATGGTGGATTTCC
416..	ATAC	80	TTTGGATGGAGCAATTCAACA	258	TTTGGATGGAGCAATTCAACatacAGA
419			GAGAATGGTGGATTTCCAT		GAATGGTGGATTTCCAT
416..	ATACA	81	TTTGGATGGAGCAATTCAACG	259	TTTGGATGGAGCAATTCAACatacaGAG
420			AGAATGGTGGATTTCCATA		AATGGTGGATTTCCATA
416..	ATACAG	82	TTTGGATGGAGCAATTCAACA	260	TTTGGATGGAGCAATTCAACatacagAG
421			GAATGGTGGATTTCCATAC		AATGGTGGATTTCCATAC
416..	ATACAGA	83	TTTGGATGGAGCAATTCAACG	261	TTTGGATGGAGCAATTCAACatacagaG
422			AATGGTGGATTTCCATACA		AATGGTGGATTTCCATACA
416..	ATACAGAGAAT	84	TTTGGATGGAGCAATTCAACG	262	TTTGGATGGAGCAATTCAACatacagaga
429	GGT (389)		GATTTCCATACACTGAAAT		atggtGGATTTCCATACACTGAAAT
417..	T	85	TTGGATGGAGCAATTCAACAA	263	TTGGATGGAGCAATTCAACAtACAGA
417			CAGAGAATGGTGGATTTCC		GAATGGTGGATTTCC
417..	TA	86	TTGGATGGAGCAATTCAACAC	264	TTGGATGGAGCAATTCAACAtaCAGA
418			AGAGAATGGTGGATTTCCA		GAATGGTGGATTTCCA
417..	TAC	87	TTGGATGGAGCAATTCAACAA	265	TTGGATGGAGCAATTCAACAtacAGAG
419			GAGAATGGTGGATTTCCAT		AATGGTGGATTTCCAT
417..	TACA	88	TTGGATGGAGCAATTCAACAG	266	TTGGATGGAGCAATTCAACAtacaGAG
420			AGAATGGTGGATTTCCATA		AATGGTGGATTTCCATA
417..	TACAG	89	TTGGATGGAGCAATTCAACAA	267	TTGGATGGAGCAATTCAACAtacagAG
421			GAATGGTGGATTTCCATAC		AATGGTGGATTTCCATAC
417..	TACAGA	90	TTGGATGGAGCAATTCAACAG	268	TTGGATGGAGCAATTCAACAtacagaGA
422			AATGGTGGATTTCCATACA		ATGGTGGATTTCCATACA
417..	TACAGAG	91	TTGGATGGAGCAATTCAACAA	269	TTGGATGGAGCAATTCAACAtacagagA
423			ATGGTGGATTTCCATACAC		ATGGTGGATTTCCATACAC
418..	AC	92	TGGATGGAGCAATTCAACATA	270	TGGATGGAGCAATTCAACATacAGAG
419			GAGAATGGTGGATTTCCAT		AATGGTGGATTTCCAT
418..	ACAG	93	TGGATGGAGCAATTCAACATA	271	TGGATGGAGCAATTCAACATacagAGA
421			GAATGGTGGATTTCCATAC		ATGGTGGATTTCCATAC
418..	ACAGAGA	94	TGGATGGAGCAATTCAACATA	272	TGGATGGAGCAATTCAACATacagagaA
424			TGGTGGATTTCCATACACT		TGGTGGATTTCCATACACT
419..	CA	95	GGATGGAGCAATTCAACATAG	273	GGATGGAGCAATTCAACATAcaGAGA
420			AGAATGGTGGATTTCCATA		ATGGTGGATTTCCATA
419..	CAG	96	GGATGGAGCAATTCAACATAA	274	GGATGGAGCAATTCAACATAcagAGA
421			GAATGGTGGATTTCCATAC		ATGGTGGATTTCCATAC
424..	AA	97	GAGCAATTCAACATACAGAGT	275	GAGCAATTCAACATACAGAGaaTGGT
425			GGTGGATTTCCATACACTG		GGATTTCCATACACTG

TABLE 5C
A list of exemplary mutant alleles obtained in the PMT2 gene.
PMT2

		Mutant		Reference
	Deleted	Allele		Allele
	sequence	Sequence		Sequence
	(SEQ	(SEQ ID	Mutant Allele	(SEQ ID
Position	ID No.)	No.)	Sequence	No.)	Reference Allele Sequence
98...98	A	98	TGGCACTTCCAAACACCA	276	TGGCACTTCCAAACACCAAAaCG
			AACGGCCACAAGAATGGG		GCCACAAGAATGGGACTT
			ACTT
196...196	T	99	CCAATTGTATTAAGCCTGG	277	CCAATTGTATTAAGCCTGGTtGG
			TGGTTTTCAGAGTTTAGCG		TTTTCAGAGTTTAGCGCA
			CA
346..350	CAACA	100	TGACTTTGGATGGAGCAAT	278	TGACTTTGGATGGAGCAATTcaac
			TCACAGAGAATGGTGGAT		aCACAGAGAATGGTGGATTTC
			TTC
346..352	CAACACA	101	TGACTTTGGATGGAGCAAT	279	TGACTTTGGATGGAGCAATTcaac
			TCAGAGAATGGTGGATTTC		acaCAGAGAATGGTGGATTTCCA
			CA
346..355	CAACACACA	102	TGACTTTGGATGGAGCAAT	280	TGACTTTGGATGGAGCAATTcaac
	G (390)		TAGAATGGTGGATTTCCAT		acacagAGAATGGTGGATTTCCATA
			AC		C
347..354	AACACACA	103	GACTTTGGATGGAGCAATT	281	GACTTTGGATGGAGCAATTCaaca
			CGAGAATGGTGGATTTCC		cacaGAGAATGGTGGATTTCCATA
			ATA
347..357	AACACACAG	104	GACTTTGGATGGAGCAATT	282	GACTTTGGATGGAGCAATTCaaca
	AG (391)		CAATGGTGGATTTCCATAC		cacagagAATGGTGGATTTCCATAC
			AC		AC
348..349	AC	105	ACTTTGGATGGAGCAATTC	283	ACTTTGGATGGAGCAATTCAacA
			AACACAGAGAATGGTGGA		CACAGAGAATGGTGGATTT
			TTT
348..351	ACAC	106	ACTTTGGATGGAGCAATTC	284	ACTTTGGATGGAGCAATTCAacac
			AACAGAGAATGGTGGATT		ACAGAGAATGGTGGATTTCC
			TCC
348..352	ACACA	107	ACTTTGGATGGAGCAATTC	285	ACTTTGGATGGAGCAATTCAacac
			ACAGAGAATGGTGGATTT		aCAGAGAATGGTGGATTTCCA
			CCA
348..353	ACACAC	108	ACTTTGGATGGAGCAATTC	286	ACTTTGGATGGAGCAATTCAacac
			AAGAGAATGGTGGATTTC		acAGAGAATGGTGGATTTCCAT
			CAT
348..354	ACACACA	109	ACTTTGGATGGAGCAATTC	287	ACTTTGGATGGAGCAATTCAacac
			AGAGAATGGTGGATTTCC		acaGAGAATGGTGGATTTCCATA
			ATA
348..355	ACACACAG	110	ACTTTGGATGGAGCAATTC	288	ACTTTGGATGGAGCAATTCAacac
			AAGAATGGTGGATTTCCAT		acagAGAATGGTGGATTTCCATAC
			AC
348..357	ACACACAGA	111	ACTTTGGATGGAGCAATTC	289	ACTTTGGATGGAGCAATTCAacac
	G (392)		AAATGGTGGATTTCCATAC		acagagAATGGTGGATTTCCATACA
			AC		C
349..349	C	112	CTTTGGATGGAGCAATTCA	290	CTTTGGATGGAGCAATTCAAcAC
			AACACAGAGAATGGTGGA		ACAGAGAATGGTGGATTT
			TTT
349..350	CA	113	CTTTGGATGGAGCAATTCA	291	CTTTGGATGGAGCAATTCAAcaC
			ACACAGAGAATGGTGGAT		ACAGAGAATGGTGGATTTC
			TTC
349..352	CACA	114	CTTTGGATGGAGCAATTCA	292	CTTTGGATGGAGCAATTCAAcaca
			ACAGAGAATGGTGGATTT		CAGAGAATGGTGGATTTCCA
			CCA
349..355	CACACAG	115	CTTTGGATGGAGCAATTCA	293	CTTTGGATGGAGCAATTCAAcaca
			AAGAATGGTGGATTTCCAT		cagAGAATGGTGGATTTCCATAC
			AC
350..351	AC	116	TTTGGATGGAGCAATTCAA	294	TTTGGATGGAGCAATTCAACacA
			CACAGAGAATGGTGGATT		CAGAGAATGGTGGATTTCC
			TCC
350..353	ACAC	117	TTTGGATGGAGCAATTCAA	295	TTTGGATGGAGCAATTCAACacac
			CAGAGAATGGTGGATTTC		AGAGAATGGTGGATTTCCAT
			CAT
350..354	ACACA	118	TTTGGATGGAGCAATTCAA	296	TTTGGATGGAGCAATTCAACacac
			CGAGAATGGTGGATTTCC		aGAGAATGGTGGATTTCCATA
			ATA
350..355	ACACAG	119	TTTGGATGGAGCAATTCAA	297	TTTGGATGGAGCAATTCAACacac
			CAGAATGGTGGATTTCCAT		agAGAATGGTGGATTTCCATAC
			AC
350..357	ACACAGAG	120	TTTGGATGGAGCAATTCAA	298	TTTGGATGGAGCAATTCAACacac
			CAATGGTGGATTTCCATAC		agagAATGGTGGATTTCCATACAC
			AC
350..363	ACACAGAGA	121	TTTGGATGGAGCAATTCAA	299	TTTGGATGGAGCAATTCAACacac
	ATGGT (393)		CGGATTTCCATACACTGAA		agagaatggtGGATTTCCATACACT
			AT		GAAAT
351..352	CA	122	TTGGATGGAGCAATTCAA	300	TTGGATGGAGCAATTCAACAcaC
			CACAGAGAATGGTGGATT		AGAGAATGGTGGATTTCCA
			TCCA
351..354	CACA	123	TTGGATGGAGCAATTCAA	301	TTGGATGGAGCAATTCAACAcaca
			CAGAGAATGGTGGATTTC		GAGAATGGTGGATTTCCATA
			CATA
351..356	CACAGA	124	TTGGATGGAGCAATTCAA	302	TTGGATGGAGCAATTCAACAcaca
			CAGAATGGTGGATTTCCAT		gaGAATGGTGGATTTCCATACA
			ACA
351..357	CACAGAG	125	TTGGATGGAGCAATTCAA	303	TTGGATGGAGCAATTCAACAcaca
			CAAATGGTGGATTTCCATA		gagAATGGTGGATTTCCATACAC
			CAC
351..362	CACAGAGAA	126	TTGGATGGAGCAATTCAA	304	TTGGATGGAGCAATTCAACAcaca
	TGG (394)		CATGGATTTCCATACACTG		gagaatggTGGATTTCCATACACTG
			AAA		AAA
352..354	ACA	127	TGGATGGAGCAATTCAAC	305	TGGATGGAGCAATTCAACACaca
			ACGAGAATGGTGGATTTC		GAGAATGGTGGATTTCCATA
			CATA
352..355	ACAG	128	TGGATGGAGCAATTCAAC	306	TGGATGGAGCAATTCAACACacag
			ACAGAATGGTGGATTTCC		AGAATGGTGGATTTCCATAC
			ATAC
353..354	CA	129	GGATGGAGCAATTCAACA	307	GGATGGAGCAATTCAACACAcaG
			CAGAGAATGGTGGATTTC		AGAATGGTGGATTTCCATA
			CATA
353..356	CAGA	130	GGATGGAGCAATTCAACA	308	GGATGGAGCAATTCAACACAcaga
			CAGAATGGTGGATTTCCAT		GAATGGTGGATTTCCATACA
			ACA
353..361	CAGAGAATG	131	GGATGGAGCAATTCAACA	309	GGATGGAGCAATTCAACACAcaga
			CAGTGGATTTCCATACACT		gaatgGTGGATTTCCATACACTGAA
			GAA
354..359	AGAGAA	132	GATGGAGCAATTCAACAC	310	GATGGAGCAATTCAACACACagag
			ACTGGTGGATTTCCATACA		aaTGGTGGATTTCCATACACTG
			CTG

TABLE 5D
A list of exemplary mutant alleles obtained in the PMT3 gene.
PMT3

		Mutant		Reference
	Deleted	Allele		Allele
	sequence	Sequence		Sequence
Pos-	(SEQ	(SEQ ID	Mutant	(SEQ ID
ition	ID No.)	No.)	Allele Sequence	No.)	Reference Allele Sequence
98...	A	133	TGGCACTTCCAAACACCAAACGGC	311	TGGCACTTCCAAACACCAAAaCGGCCA
98			CACCAGAATGGCACTT		CCAGAATGGCACTT
280...	T	134	CCAACTCTATTAAGCCTGGTGGTTT	312	CCAACTCTATTAAGCCTGGTtGGTTTTC
280			TCAGAGTTTAGCGCA		AGAGTTTAGCGCA
413..	CT	135	AACATATGGGAAGGTTCTGATTGG	313	AACATATGGGAAGGTTCTGActTTGGAT
414			ATGGAGCAATTCAACA		GGAGCAATTCAACA
418..	GA	136	ATGGGAAGGTTCTGACTTTGTGGA	314	ATGGGAAGGTTCTGACTTTGgaTGGAGC
419			GCAATTCAACACACAG		AATTCAACACACAG
426..	AA	137	GTTCTGACTTTGGATGGAGCTTCA	315	GTTCTGACTTTGGATGGAGCaaTTCAAC
427			ACACACAGAGAATGGT		ACACAGAGAATGGT
429..	TCAACAC	138	CTGACTTTGGATGGAGCAATACAG	316	CTGACTTTGGATGGAGCAATtcaacacACA
435			AGAATGGTGGATTTCC		GAGAATGGTGGATTTCC
429..	TCAACACACA	139	CTGACTTTGGATGGAGCAATGAGA	317	CTGACTTTGGATGGAGCAATtcaacacaca
438	(395)		ATGGTGGATTTCCATA		GAGAATGGTGGATTTCCATA
429..	TCAACACACA	140	CTGACTTTGGATGGAGCAATAGAA	318	CTGACTTTGGATGGAGCAATtcaacacaca
439	G (396)		TGGTGGATTTCCATAC		gAGAATGGTGGATTTCCATAC
430..	CAACACA	141	TGACTTTGGATGGAGCAATTCAGA	319	TGACTTTGGATGGAGCAATTcaacacaCAG
436			GAATGGTGGATTTCCA		AGAATGGTGGATTTCCA
431..	A	142	GACTTTGGATGGAGCAATTCACAC	320	GACTTTGGATGGAGCAATTCaACACACA
431			ACAGAGAATGGTGGAT		GAGAATGGTGGAT
431..	AA	143	GACTTTGGATGGAGCAATTCCACA	321	GACTTTGGATGGAGCAATTCaaCACACA
432			CAGAGAATGGTGGATT		GAGAATGGTGGATT
431..	AACACACA	144	GACTTTGGATGGAGCAATTCGAGA	322	GACTTTGGATGGAGCAATTCaacacacaGA
438			ATGGTGGATTTCCATA		GAATGGTGGATTTCCATA
431..	AACACACAGA	145	GACTTTGGATGGAGCAATTCAATG	323	GACTTTGGATGGAGCAATTCaacacacaga
441	G (397)		GTGGATTTCCATACAC		gAATGGTGGATTTCCATACAC
432..	AC	146	ACTTTGGATGGAGCAATTCAACAC	324	ACTTTGGATGGAGCAATTCAacACACAG
433			AGAGAATGGTGGATTT		AGAATGGTGGATTT
432..	ACAC	147	ACTTTGGATGGAGCAATTCAACAG	325	ACTTTGGATGGAGCAATTCAacacACAGA
435			AGAATGGTGGATTTCC		GAATGGTGGATTTCC
432..	ACACAC	148	ACTTTGGATGGAGCAATTCAAGAG	326	ACTTTGGATGGAGCAATTCAacacacAGA
437			AATGGTGGATTTCCAT		GAATGGTGGATTTCCAT
432..	ACACACAG	149	ACTTTGGATGGAGCAATTCAAGAA	327	ACTTTGGATGGAGCAATTCAacacacagAG
439			TGGTGGATTTCCATAC		AATGGTGGATTTCCATAC
432..	ACACACAGAG	150	ACTTTGGATGGAGCAATTCAAATG	328	ACTTTGGATGGAGCAATTCAacacacagag
441	(398)		GTGGATTTCCATACAC		AATGGTGGATTTCCATACAC
432..	ACACACAGAG	151	ACTTTGGATGGAGCAATTCATGGA	329	ACTTTGGATGGAGCAATTCAacacacagag
446	AATGG(399)		TTTCCATACACTGAAA		aatggTGGATTTCCATACACTGAAA
432..	ACACACAGAG	152	ACTTTGGATGGAGCAATTCAGATT	330	ACTTTGGATGGAGCAATTCAacacacagag
448	AATGGTG		TCCATACACTGAAATG		aatggtgGATTTCCATACACTGAAATG
	(400)
433..	C	153	CTTTGGATGGAGCAATTCAAACAC	331	CTTTGGATGGAGCAATTCAAcACACAG
433			AGAGAATGGTGGATTT		AGAATGGTGGATTT
433..	CACA	154	CTTTGGATGGAGCAATTCAACAGA	332	CTTTGGATGGAGCAATTCAAcacaCAGAG
436			GAATGGTGGATTTCCA		AATGGTGGATTTCCA
433..	CACAC	155	CTTTGGATGGAGCAATTCAAAGAG	333	CTTTGGATGGAGCAATTCAAcacacAGAG
437			AATGGTGGATTTCCAT		AATGGTGGATTTCCAT
433..	CACACAG	156	CTTTGGATGGAGCAATTCAAAGAA	334	CTTTGGATGGAGCAATTCAAcacacagAGA
439			TGGTGGATTTCCATAC		ATGGTGGATTTCCATAC
434..	AC	157	TTTGGATGGAGCAATTCAACACAG	335	TTTGGATGGAGCAATTCAACacACAGAG
435			AGAATGGTGGATTTCC		AATGGTGGATTTCC
434..	ACAC	158	TTTGGATGGAGCAATTCAACAGAG	336	TTTGGATGGAGCAATTCAACacacAGAG
437			AATGGTGGATTTCCAT		AATGGTGGATTTCCAT
434..	ACACA	159	TTTGGATGGAGCAATTCAACGAGA	337	TTTGGATGGAGCAATTCAACacacaGAGA
438			ATGGTGGATTTCCATA		ATGGTGGATTTCCATA
435..	CA	160	TTGGATGGAGCAATTCAACACAGA	338	TTGGATGGAGCAATTCAACAcaCAGAGA
436			GAATGGTGGATTTCCA		ATGGTGGATTTCCA
435..	CACA	161	TTGGATGGAGCAATTCAACAGAGA	339	TTGGATGGAGCAATTCAACAcacaGAGA
438			ATGGTGGATTTCCATA		ATGGTGGATTTCCATA
435..	CACAG	162	TTGGATGGAGCAATTCAACAAGAA	340	TTGGATGGAGCAATTCAACAcacagAGAA
439			TGGTGGATTTCCATAC		TGGTGGATTTCCATAC
435..	CACAGAGAAT	163	TTGGATGGAGCAATTCAACAGGAT	341	TTGGATGGAGCAATTCAACAcacagagaat
447	GGT (401)		TTCCATACACTGAAAT		ggtGGATTTCCATACACTGAAAT
436..	AC	164	TGGATGGAGCAATTCAACACAGAG	342	TGGATGGAGCAATTCAACACacAGAGA
437			AATGGTGGATTTCCAT		ATGGTGGATTTCCAT
436..	ACAG	165	TGGATGGAGCAATTCAACACAGAA	343	TGGATGGAGCAATTCAACACacagAGAA
439			TGGTGGATTTCCATAC		TGGTGGATTTCCATAC
436..	ACAGAGAA	166	TGGATGGAGCAATTCAACACTGGT	344	TGGATGGAGCAATTCAACACacagagaaTG
443			GGATTTCCATACACTG		GTGGATTTCCATACACTG
437..	CA	167	GGATGGAGCAATTCAACACAGAG	345	GGATGGAGCAATTCAACACAcaGAGAA
438			AATGGTGGATTTCCATA		TGGTGGATTTCCATA
440..	AGA	168	TGGAGCAATTCAACACACAGATGG	346	TGGAGCAATTCAACACACAGagaATGGT
442			TGGATTTCCATACACT		GGATTTCCATACACT
440..	AGAA	169	TGGAGCAATTCAACACACAGTGGT	347	TGGAGCAATTCAACACACAGagaaTGGT
443			GGATTTCCATACACTG		GGATTTCCATACACTG
441..	G	170	GGAGCAATTCAACACACAGAAATG	348	GGAGCAATTCAACACACAGAgAATGGT
441			GTGGATTTCCATACAC		GGATTTCCATACAC
445..	GGTGG	171	CAATTCAACACACAGAGAATATTT	349	CAATTCAACACACAGAGAATggtggATTT
449			CCATACACTGAAATGA		CCATACACTGAAATGA
451..	TTTCCATACA	172	AACACACAGAGAATGGTGGAAAA	350	AACACACAGAGAATGGTGGAtttccataca
463	CTG (402)		TGATTGTTCATCTTCCA		ctgAAATGATTGTTCATCTTCCA

TABLE 5E
A list of exemplary mutant alleles obtained in the PMT4 gene.
PMT4

		Mutant		Reference
	Deleted	Allele		Allele
	sequence	Sequence		Sequence
Pos-	(SEQ ID	(SEQ ID		(SEQ ID
ition	No.)	No.)	Mutant Allele Sequence	No.)	Reference Allele Sequence
131...	A	173	CGGCACTTCCAAACACCAAACGGCCA	351	CGGCACTTCCAAACACCAAAaCGGCCAC
131			CCATAATGGCACTT		CATAATGGCACTT
541..	ATTCAACACA	174	TTTTGACTTTGGATGGAGCAAGAGAAT	352	TTTTGACTTTGGATGGAGCAattcaacacacA
551	C (403)		GGTGGATTTCCAT		GAGAATGGTGGATTTCCAT
543..	TCAACACACA	175	TTGACTTTGGATGGAGCAATGAATGGT	353	TTGACTTTGGATGGAGCAATtcaacacacaga
554	GA (404)		GGATTTCCATACA		GAATGGTGGATTTCCATACA
544..	CAACACA	176	TGACTTTGGATGGAGCAATTCAGAGA	354	TGACTTTGGATGGAGCAATTcaacacaCAG
550			ATGGTGGATTTCCA		AGAATGGTGGATTTCCA
544..	CAACACACAG	177	TGACTTTGGATGGAGCAATTAGAATGG	355	TGACTTTGGATGGAGCAATTcaacacacagA
553	(405)		TGGATTTCCATAC		GAATGGTGGATTTCCATAC
545..	AACACACAGA	178	GACTTTGGATGGAGCAATTCAATGGTG	356	GACTTTGGATGGAGCAATTCaacacacagag
555	G (406)		GATTTCCATACAC		AATGGTGGATTTCCATACAC
545..	AACACACAGA	179	GACTTTGGATGGAGCAATTCTGGTGGA	357	GACTTTGGATGGAGCAATTCaacacacagaga
557	GAA (407)		TTTCCATACACTG		aTGGTGGATTTCCATACACTG
546..	AC	180	ACTTTGGATGGAGCAATTCAACACAG	358	ACTTTGGATGGAGCAATTCAacACACAG
547			AGAATGGTGGATTT		AGAATGGTGGATTT
546..	ACAC	181	ACTTTGGATGGAGCAATTCAACAGAG	359	ACTTTGGATGGAGCAATTCAacacACAGA
549			AATGGTGGATTTCC		GAATGGTGGATTTCC
546..	ACACAC	182	ACTTTGGATGGAGCAATTCAAGAGAA	360	ACTTTGGATGGAGCAATTCAacacacAGAG
551			TGGTGGATTTCCAT		AATGGTGGATTTCCAT
546..	ACACACA	183	ACTTTGGATGGAGCAATTCAGAGAAT	361	ACTTTGGATGGAGCAATTCAacacacaGAG
552			GGTGGATTTCCATA		AATGGTGGATTTCCATA
546..	ACACACAG	184	ACTTTGGATGGAGCAATTCAAGAATG	362	ACTTTGGATGGAGCAATTCAacacacagAG
553			GTGGATTTCCATAC		AATGGTGGATTTCCATAC
547..	CACA	185	CTTTGGATGGAGCAATTCAACAGAGA	363	CTTTGGATGGAGCAATTCAAcacaCAGAG
550			ATGGTGGATTTCCA		AATGGTGGATTTCCA
547..	CACAC	186	CTTTGGATGGAGCAATTCAAAGAGAA	364	CTTTGGATGGAGCAATTCAAcacacAGAG
551			TGGTGGATTTCCAT		AATGGTGGATTTCCAT
548..	AC	187	TTTGGATGGAGCAATTCAACACAGAG	365	TTTGGATGGAGCAATTCAACacACAGAG
549			AATGGTGGATTTCC		AATGGTGGATTTCC
548..	ACAC	188	TTTGGATGGAGCAATTCAACAGAGAA	366	TTTGGATGGAGCAATTCAACacacAGAGA
551			TGGTGGATTTCCAT		ATGGTGGATTTCCAT
548..	ACACA	189	TTTGGATGGAGCAATTCAACGAGAAT	367	TTTGGATGGAGCAATTCAACacacaGAGA
552			GGTGGATTTCCATA		ATGGTGGATTTCCATA
549..	CACA	190	TTGGATGGAGCAATTCAACAGAGAAT	368	TTGGATGGAGCAATTCAACAcacaGAGAA
552			GGTGGATTTCCATA		TGGTGGATTTCCATA
549..	CACAG	191	TTGGATGGAGCAATTCAACAAGAATG	369	TTGGATGGAGCAATTCAACAcacagAGAA
553			GTGGATTTCCATAC		TGGTGGATTTCCATAC
550..	AC	192	TGGATGGAGCAATTCAACACAGAGAA	370	TGGATGGAGCAATTCAACACacAGAGAA
551			TGGTGGATTTCCAT		TGGTGGATTTCCAT
550..	ACA	193	TGGATGGAGCAATTCAACACGAGAAT	371	TGGATGGAGCAATTCAACACacaGAGAA
552			GGTGGATTTCCATA		TGGTGGATTTCCATA
550..	ACAG	194	TGGATGGAGCAATTCAACACAGAATG	372	TGGATGGAGCAATTCAACACacagAGAAT
553			GTGGATTTCCATAC		GGTGGATTTCCATAC
550..	ACAGAGA	195	TGGATGGAGCAATTCAACACATGGTG	373	TGGATGGAGCAATTCAACACacagagaATG
556			GATTTCCATACACT		GTGGATTTCCATACACT
551..	CA	196	GGATGGAGCAATTCAACACAGAGAAT	374	GGATGGAGCAATTCAACACAcaGAGAAT
552			GGTGGATTTCCATA		GGTGGATTTCCATA
554..	A	197	TGGAGCAATTCAACACACAGGAATGG	375	TGGAGCAATTCAACACACAGaGAATGGT
554			TGGATTTCCATACA		GGATTTCCATACA
558..	TGGTGG	198	GCAATTCAACACACAGAGAAATTTCC	376	GCAATTCAACACACAGAGAAtggtggATTT
563			ATACACTGAAATGA		CCATACACTGAAATGA
565..	TT	199	AACACACAGAGAATGGTGGATCCATA	377	AACACACAGAGAATGGTGGAttTCCATAC
566			CACTGAAATGATTG		ACTGAAATGATTG
569..	CATA	200	CACAGAGAATGGTGGATTTCCACTGA	378	CACAGAGAATGGTGGATTTCcataCACTG
572			AATGATTGTTCATC		AAATGATTGTTCATC

Example 3: Alkaloid Analysis of PMT Edited Lines

Genome edited tobacco plants along with controls are grown in 10″ pots in green house with 75 PPM fertilizer. At flowering stage, plants are topped and 2 weeks post topping lamina samples were collected from 3, 4, 5 leaves from top and alkaloid levels are measured (Tables 6A to 6C) using a method in accordance with CORESTA Method No 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and those defined in the Centers for Disease Control and Prevention's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, as published in the Federal Register Vol. 64, No. 55 Mar. 23, 1999 (and as amended in Vol. 74, No. 4, Jan. 7, 2009).

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 %) on either an as is or dry weight basis. Reporting data on a dry weight basis requires an oven volatiles (OV) determination. Unless specified otherwise, total or individual alkaloid levels or nicotine levels shown herein are on a dry weight basis (e.g., percent total alkaloid or percent nicotine).

Plants are also planted in the field, harvested, and tested for alkaloids and TSNA levels in cured tobacco. Both leaf yield and leaf grade are also assessed for PMT edited plants. Further, different mutant combinations of individual PMT genes are generated and tested (e.g., single, double, triple, or quadruple mutants).

Example 4: Comparing a Quintuple Pmt Knock-Out Mutant with Other Low-Alkaloid Tobacco Plants

A quintuple pmt knock-out mutant line CS15 (see Table 4E for genotype, in the NLM (Ph Ph) background) is grown side by side with a PMT RNAi transgenic line (in the VA359 background, as described in US 2015/0322451) and a low-nicotine KY171 (“LN KY171”) variety (the KY 171 background harboring nic1 and nic2 double mutations). Leaves are harvested and cured via a dark fire curing method. Each line is analyzed for nicotine and total alkaloid levels, leaf yield, and leaf quality (FIGS. 2 to 5). The data shows that suppressing PMT gene activity by editing all five PMT genes reduces nicotine level without comprising leaf yield or quality.

Example 5: Obtaining Tobacco Lines with Edited Mutant Alleles in One or More PMT Genes

Tobacco lines with mutations in individual PMT genes or selected combinations of PMT genes are obtained from the tobacco lines listed in Table 3. Crossing a quintuple, quadruple, triple, or double mutant (having mutations in five, four, three, or two PMT genes, respectively) to a non-mutated control line and selecting segregating progeny plants for specific PMT mutation combinations. Tables 7A to 7E represents possible mutant combinations being obtained. Each mutated gene can be either homozygous or heterozygous for the mutation. Each of the mutant alleles listed in Tables 4A to 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 to 9E.

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 related to alkaloid biosynthesis in tobacco, such as quinolate phosphoribosyl transferase (QPT) or quinolinate synthase (QS). Briefly, gene editing is used to mutant selected QPT and/or QS genes in a desired pmt mutant background (e.g., a quadruple or quintuple pmt mutant). In the resulting combined qpt/pmt or qs/pmt mutants, alkaloids and TSNA levels are tested in cured tobacco. Both leaf yield and leaf grade are also assessed.

TABLE 6A
Alkaloid levels in PMT edited lines in K326 (shown
here and Tables 6B, 6C, and 7 as weight percentage
per gram leaf lamina (dry weight))

% Alkaloids

	Variety	Plant ID	Nicotine	Total Alkaloids

	K326	17GH1811	1.17	1.23
	Control	17GH1822	1.63	1.71
		17GH1806	1.69	1.76
		17GH1899	1.7893	1.9194
		17GH1812	1.91	2
		17GH1900	2.088	2.239
		17GH1821	2.16	2.26
		17GH1896	2.6006	2.7359
	K326	17GH1810	0.0013	0.3
	Edited	17GH1808	0.0044	0.24
		17GH1901	0.006	0.6958
		17GH1893	0.0072	0.7351
		17GH1804	0.0078	0.44
		17GH1902	0.008	0.6245
		18GH4	0.0102	0.2688
		17GH1892	0.0209	0.1281

TABLE 6B
Alkaloid levels in PMT edited lines in TN90

% Alkaloids

	Variety	Plant ID	Nicotine	Total Alkaloids

	TN90	17GH1838	1.88	1.98
	Control	17GH1923	2.0868	2.2136
		17GH1924	2.2099	2.3394
		17GH1718	2.29	2.42
		17GH1839	2.6	2.74
		17GH1909	2.7639	2.9429
		17GH1910	2.9346	3.1283
	TN90	17GH1699	0.0011	0.58
	Edited	17GH1708	0.0014	0.56
		17GH1847	0.0016	0.6
		17GH1848	0.0018	0.42
		17GH1724	0.0022	0.59
		17GH1846	0.0022	0.41
		17GH1722	0.0023	0.62
		17GH1725	0.003	0.69
		17GH1717	0.0035	0.7
		17GH1719	0.0042	0.75
		17GH1845	0.0047	0.45
		17GH1943	0.007	0.3464
		18GH47	0.0072	1.0455
		17GH1944	0.0074	0.403
		17GH1932	0.0074	0.4758
		17GH1936	0.0074	1.4394
		17GH1918	0.0075	0.458
		17GH1912	0.0078	0.5234
		18GH31	0.0079	1.0902
		18GH28	0.008	0.8748
		17GH1928	0.0081	1.1024
		17GH1933	0.0083	0.6517
		17GH1911	0.0088	0.281

TABLE 6C
Alkaloid levels in PMT edited lines in Narrow Leaf Madole (NLM)

% Alkaloids

	Variety	Plant ID	Nicotine	Total Alkaloids

	NLM	18GH126	2.0844	2.1734
	Control	18GH7	3.3504	3.5136
	NLM	18GH10	0.001	1.14
	Edited	18GH9	0.0012	0.92
		18GH6	0.0019	1.46
		18GH8	0.0022	1.46
		17GH1905	0.0032	1.49
		18GH5	0.0038	0.92
		18GH130	0.0041	0.8756
		18GH132	0.0044	0.6335
		18GH79	0.0045	0.6182
		18GH69	0.0069	0.7495
		18GH71	0.007	0.7726
		18GH131	0.0077	0.4289
		18GH66	0.0081	0.6951
		18GH227	0.0086	0.8726
		18GH78	0.0086	0.662
		18GH72	0.0087	1.0048
		18GH216	0.0089	1.2758
		18GH65	0.0094	0.7018

TABLE 7
Relative changes in nicotine and total alkaloid levels in quintuple
pmt knock-out mutants in various varieties. Average percent
levels of nicotine and total alkaloids are calculated based
on percent level data from individual lines as shown in Tables
6A to 6C. Relative changes reflect the nicotine or total alkaloid
level in a quintuple pmt mutant relative to its control.

	Nicotine	Total Alkaloids

	K326 Control	1.880	1.982
	K326 quintuple	0.008	0.429
	pmt mutant
	Relative change	0.44%	21.65%
	TN90 Control	2.395	2.538
	TN90 quintuple	0.005	0.655
	pmt mutant
	Relative change	0.22%	25.80%
	NLM Control	2.717	2.844
	NLM quintuple	0.006	0.927
	pmt mutant
	Relative change	0.20%	32.59%

TABLE 8A
A list of mutants obtained with various genotypic combinations
for five PMT genes: single gene mutations

	PMT1a	PMT1b	PMT2	PMT3	PMT4

1	Mutant	WT	WT	WT	WT
2	WT	Mutant	WT	WT	WT
3	WT	WT	Mutant	WT	WT
4	WT	WT	WT	Mutant	WT
5	WT	WT	WT	WT	Mutant

TABLE 8B
A list of mutants obtained with various genotypic combinations
for five PMT genes: double gene mutations

	PMT1a	PMT1b	PMT2	PMT3	PMT4

1	Mutant	Mutant	WT	WT	WT
2	Mutant	WT	Mutant	WT	WT
3	Mutant	WT	WT	Mutant	WT
4	Mutant	WT	WT	WT	Mutant
5	WT	Mutant	Mutant	WT	WT
6	WT	Mutant	WT	Mutant	WT
7	WT	Mutant	WT	WT	Mutant
8	WT	WT	Mutant	Mutant	WT
9	WT	WT	Mutant	WT	Mutant
10	WT	WT	WT	Mutant	Mutant

TABLE 8C
A list of mutants obtained with various genotypic combinations
for five PMT genes: triple gene combinations

	PMT1a	PMT1b	PMT2	PMT3	PMT4

1	Mutant	Mutant	Mutant	WT	WT
2	Mutant	Mutant	WT	Mutant	WT
3	Mutant	Mutant	WT	WT	Mutant
4	Mutant	WT	Mutant	Mutant	WT
5	Mutant	WT	Mutant	WT	Mutant
6	Mutant	WT	WT	Mutant	Mutant
7	WT	Mutant	Mutant	Mutant	WT
8	WT	Mutant	Mutant	WT	Mutant
9	WT	WT	Mutant	Mutant	Mutant
10	WT	Mutant	Mutant	WT	Mutant

TABLE 8D
A list of mutants obtained with various genotypic combinations
for five PMT genes: quadruple gene combinations

	PMT1a	PMT1b	PMT2	PMT3	PMT4

1	Mutant	Mutant	Mutant	Mutant	WT
2	WT	Mutant	Mutant	Mutant	Mutant
3	Mutant	WT	Mutant	Mutant	Mutant
4	Mutant	Mutant	WT	Mutant	Mutant
5	Mutant	Mutant	Mutant	WT	Mutant

TABLE 8E
A list of mutants obtained with various genotypic combinations
for five PMT genes: quintuple gene combinations

	PMT1a	PMT1b	PMT2	PMT3	PMT4

1	Mutant	Mutant	Mutant	Mutant	Mutant

Example 7: PMT Genome Editing and Tobacco Line Development

Additional PMT knockout mutants are produced by editing all five PMT genes (PMT1a, PMT1b, PMT2, PMT3, and PMT4) in different tobacco lines. Tobacco protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding a a genome editing technology (GET2) protein and specific guide RNAs (gRNAs) targeting PMT genes at desired positions. Table 9 lists gRNA sequences used for PMT editing. Some gRNAs (e.g., Nos. 6 and 7) are pooled together for targeting multiple PMT genes in a single transfection.

TABLE 9
Guide RNAs for GET2 used in Example 7.
“Y” indicates that a gRNA is capable of
targeting that PMT gene, while represents
that a gRNA does not target that PMT gene.

gRNA Sequence	PMT1a	PMT1b	PMT2	PMT3	PMT4
GATGGAGCAATTCAACATACAGA	Y	Y	—	—	—
(SEQ ID NO: 730)
GATGGAGCAATTCAACACACAGA	—	—	Y	Y	Y
(SEQ ID NO: 731)

Transfected protoplasts are then immobilized in 1% agarose bead and subjected to tissue culture. When calli grow up to ˜1 mm in diameter, they are spread on TOM2 plates. Calli are screened for insertions or deletions (indels) at the target positions using fragment analysis. Candidates, showing size shifts compared to wildtype control, are selected for further culture and the consequent shoots are tested by fragment analysis again to confirm the presence of indels. Rooted shoots are potted and sequenced for the target positions to determine the exact sequences deleted. Young leaf from each plant is harvested and PCR amplified for PMT fragments using phirekit. PMT Libraries for each line is indexed and 384 lines are pooled and sequenced using Miseq.

SNP analysis is carried out to determine both the exact edited pmt mutant allele sequences and the zygosity state at each PMT gene locus. Table 10 provides indels sequence information in each edited line of various tobacco varieties (e.g., Basma, K326, Katerini, TN90, Izmir).

TABLE 10
Mutant pmt alleles in various lines produced by genome editing using GET2. The
position of each edited site (e.g., indels) 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 PMTT SEQ ID NO: 10 for PMT4).
SEQ ID Numbers are assigned and shown for sequences of more than 10 nucleotides.

PMT1a

PMT1b

PMT2

PMT3

PMT4

Geno		Deleted	Posi-	Deleted	Posi-	Deleted	Posi-	Deleted	Posi-	Deleted	Posi-
type	Line	Sequence	tion	Sequence	tion	Sequence	tion	Sequence	tion	Sequence	tion
BAS	CS107	CAACATA	412 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAC	432 . . .	ACAC	546 . . .
MA			418				349		435		549
BAS	CS106	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAC	432 . . .	ACAC	546 . . .
MA			417				349		435		549
K326	CS115	TCAACA	411 . . .	ACAT	414 . . . 417	AC	348 . . .	CACA	433 . . .	ACACA	548 . . .
		TACA	420				349	C	437		552
		(SEQ ID
		NO: 379)
K326	18GH	ACAT	414 . . .	ACAT	414 . . . 417	ACAC	348 . . .	AC	432 . . .	ACACA	548 . . .
	2162		417			ACAG	355		433		552
K326	CS111	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAC	432 . . .	CACAC	547 . . .
			417				349		435		551
K326	CS112	CATACAG	415 . . .	AC	418 . . . 419	AC	348 . . .	ACAC	432 . . .	CACAC	547 . . .
			421				349		435		551
K326	17GH	CATACAG	415 . . .	AC	418 . . . 419	AC	348 . . .	ACAC	432 . . .	ACAC	546 . . .
	1678-		421				349		435		549
	60
K326	CS131	ACAT	414 . . .	ACAT	414 . . . 417	ACAC	348 . . .	AACA	431 . . .	ACAC	546 . . .
			417				351	CACA	439		549
								G
KAT	CS164	AT	416 . . .	CAACA	412 . . . 418	AC	348 . . .	ACAC	432 . . .	AC	546 . . .
ERIN			417	TA			349		435		547
I
KAT	CS163	ACAT	414 . . .	AT	416 . . . 417	AC	348 . . .	ACAC	432 . . .	AC	546 . . .
ERIN			417				349		435		547
I
KAT	CS146	GAGCAA	404 . . .	ACAT	414 . . . 417	AC	348 . . .	CAAC	430 . . .	AC	546 . . .
ERIN		TTCAAC	422				349	ACA	436		547
I		ATACAG
		A
		(SEQ ID
		NO: 408)
KAT	CS147	ACAT	414 . . .	CAACA	412 . . . 418	AC	348 . . .	CAAC	430 . . .	AC	546 . . .
ERIN			417	TA			349	ACA	436		547
I
KAT	CS150	AT	416 . . .	ACAT	414 . . . 417	AC	348 . . .	CACA	433 . . .	AC	546 . . .
ERIN			417				349	CAG	439		547
I
KAT	CS151	AT	416 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAC	432 . . .	CAACA	544 . . .
ERIN			417				349		435	CACAG	553
I										(SEQ ID
										NO:
										390)
KAT	CS148	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	CACA	433 . . .	AC	546 . . .
ERIN			417				349	CAG	439		547
I
KAT	CS149	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAC	432 . . .	CAACA	544 . . .
ERIN			417				349		435	CACAG	553
I										(SEQ ID
										NO:
										390)
KAT	CS152	AC	418 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAC	432 . . .	ACAC	546 . . .
ERIN			419				349		435		549
I
KAT	CS153	CAACATA	412 . . .	AC	414 . . . 415	AC	348 . . .	ACAC	432 . . .	ACAC	546 . . .
ERIN			418				349		435		549
I
KAT	CS102	ACAT	414 . . .	AACAT	413 . . . 417	ACAC	348 . . .	AC	432 . . .	AC	546 . . .
ERIN			417			ACAG	355		433		547
I
KAT	CS103	AC	418 . . .	CAACA	412 . . . 418	ACAC	348 . . .	AC	432 . . .	AC	546 . . .
ERIN			419	TA		ACAG	355		433		547
I
TN90	CS143	TACAGAG	417 . . .	ACAT	414 . . . 417	AC	348 . . .	AC	432 . . .	ACAC	546 . . .
			423				349		433		549
TN90	18GH	AC	418 . . .	ACAT	414 . . . 417	ACAC	348 . . .	ACAC	432 . . .	AC	546 . . .
	2169		419				351		435		547
TN90	CS120	ACAT	414 . . .	ACAG	418 . . . 421	ACAC	348 . . .	AC	432 . . .	AC	546 . . .
			417				351		433		547
TN90	17GH	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	ACAG	436 . . .	AC	546 . . .
	1698-		417				349		439		547
	22
TN90	17GH	ACAT	414 . . .	AACAT	413 . . . 417	AC	348 . . .	AC	432 . . .	ACAC	546 . . .
	1700-		417				349		433		549
	13
TN90	17GH	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	AC	432 . . .	CACAG	549 . . .
	1702-		417				349		433		553
	17
TN90	18GH	ACAT	414 . . .	ACAT	414 . . . 417	ACAC	348 . . .	CAAC	430 . . .	AC	546 . . .
	2171		417				351	ACA	436		547
TN90	CS165	ACAT	414 . . .	ACAT	414 . . . 417	ACAC	348 . . .	ACAC	432 . . .	ACAC	546 . . .
			417				351		435		549
TN90	CS118	ACAT	414 . . .	ACAT	414 . . . 417	AC	348 . . .	AC	432 . . .	ACAC	546 . . .
			417				349		433		549
TN90	CS133	GGAGC	403 . . .	CAACA	412 . . . 421	ACAC	348 . . .	AC	432 . . .	AC	546 . . .
		AATTC	415	TACAG			351		433		547
		AAC		(SEQ ID
		(SEQ ID		NO:
		NO: 409)		380)
TN90	17GH	CA	415 . . .	ACAT	414 . . . 417	ACAC	348 . . .	AC	432 . . .	AC	546 . . .
	1737-		416				351		433		547
	24
IZMI	18GH	CAACATA	412 . . .	ATAGA	416 . . . 417	ACAC	348 . . .	ACAC	432 . . .	ACACA	546 . . .
R	2254-		418	GAA	&		351		435	CAG	553
	7				420 . . . 425

Table 11 provides the length (in nucleotides) of each PMT indel for each gene in each line as provided in Table 10.

TABLE 11
The length (in nucleotides) of each indel for selected lines provided in Table 10.

Genotype	Line	Seed Ids	PMT1a	PMT1b	PMT2	PMT3	PMT4

BASMA	CS107	CS107	7	4	2	4	4
BASMA	CS106	CS106	4	4	2	4	4
K326	CS115	CS115	10	4	2	5	5
K326	17GH1809-13	18GH2162	4	4	8	2	5
K326	CS111	CS111	4	4	2	4	5
K326	CS112	CS112	7	2	2	4	5
K326	17GH1678-60	17GH1678-60	7	2	2	4	4
K326	CS131	CS131	4	4	4	9	4
KATERINI	18GH709-01	CS164	2	7	2	4	2
KATERINI	18GH709-08	CS163	4	2	2	4	2
KATERINI	18GH414-11	CS146	19	4	2	7	2
KATERINI	18GH414-19	CS147	4	7	2	7	2
KATERINI	18GH437-04	CS150	2	4	2	7	2
KATERINI	18GH437-08	CS151	2	4	2	4	10
KATERINI	18GH437-32	CS148	4	4	2	7	2
KATERINI	18GH437-39	CS149	4	4	2	4	10
KATERINI	18GH449-26	CS152	2	4	2	4	4
KATERINI	18GH449-33	CS153	7	2	2	4	4
KATERINI	18GH125-48	CS162	2	7	8	2	2
KATERINI	CS102	CS102	4	5	8	2	2
KATERINI	CS103	CS103	2	7	8	2	2
TN90	17GH1719-30	CS143	7	4	2	2	4
TN90	17GH1740-36	18GH2169	2	4	4	4	2
TN90	17GH1698-22	17GH1698-22	4	4	2	4	2
TN90	17GH1700-13	17GH1700-13	4	5	2	2	4
TN90	17GH1702-17	17GH1702-17	4	4	2	2	5
TN90	17GH1849-01	18GH2171	4	4	4	7	2
TN90	17GH1849-48	CS165	4	4	4	4	4
TN90	17GH1737-24	17GH1737-24	2	4	4	2	2
TN90	CS118	CS118	4	4	2	2	4
TN90	CS133	CS133	13	10	4	2	2
TN90	CS120	CS120	4	4	4	2	2
IZMIR	18GH1108-07	18GH2254-7	7	8	4	4	8

Tables 12A to 12E provide genomic sequences of approximately 90 nucleotides from each pmt mutant allele with the edited site in the middle of the genomic sequence (e.g., 45 nucleotides on each side of the deleted or inserted sequence site).

TABLE 12A
A list of exemplary mutant alleles obtained
in the PMTla gene. Mutant allele sequences
listed here represent approximately 90-
nucleotide-long genomic sequences from each
edited PMT1a gene with the edited site in
the middle of the genomic sequence (e.g.,
45 nucleotides on each side of the deleted
sequence site). The mutant allele corresponds
to the indel provided for each line in Table 10.
The lowercase letters in the reference allele
sequence (SEQ ID NO: 6) denote which nucleotides
are deleted in the mutant allele.

			Mutant		Reference
			Allele	Reference	Allele
Geno-		Mutant Allele	SEQ	Allele	SEQ
type	Line	Sequence	ID NO.	Sequence	ID NO.
BASMA	CS107	TCAGCAACTTATGGG	410	TCAGCAACTTATGGG	442
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		GAGAATGGTGGATTT		catacaGAGAATGGT
		CCATACACTGAAATG		GGATTTCCATACACT
		ATTGTTCATCTA		GAAATGATTGTTCAT
				CTA
BASMA	CS106	TCAGCAACTTATGGG	411	TCAGCAACTTATGGG	443
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	CS115	TCAGCAACTTATGGG	412	TCAGCAACTTATGGG	444
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATGAG		GATGGAGCAATtcaaca
		AATGGTGGATTTCCA		tacaGAGAATGGTGGA
		TACACTGAAATGATT		TTTCCATACACTGAA
		GTTCATCTA		ATGATTGTTCATCTA
K326	18GH	TCAGCAACTTATGGG	413	TCAGCAACTTATGGG	445
	2162	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	CS111	TCAGCAACTTATGGG	414	TCAGCAACTTATGGG	446
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	CS112	TCAGCAACTTATGGG	415	TCAGCAACTTATGGG	447
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		AAGAATGGTGGATTT		AcatacagAGAATGGTG
		CCATACACTGAAATG		GATTTCCATACACTG
		ATTGTTCATCTA		AAATGATTGTTCATC
				TATCAAAGAATGG
K326	17GH	TCAGCAACTTATGGG	416	TCAGCAACTTATGGG	448
	1678-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	60	GATGGAGCAATTCA		GATGGAGCAATTCA
		AAGAATGGTGGATTT		AcatacagAGAATGGTG
		CCATACACTGAAATG		GATTTCCATACACTG
		ATTGTTCATCTA		AAATGATTGTTCATC
				TATCAAAGAATGG
K326	CS131	TCAGCAACTTATGGG	417	TCAGCAACTTATGGG	449
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS164	TCAGCAACTTATGGG	418	TCAGCAACTTATGGG	450
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACACAGAGAATGGT		ACatACAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
KATERI	CS163	TCAGCAACTTATGGG	419	TCAGCAACTTATGGG	451
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS146	TCAGCAACTTATGGG	420	TCAGCAACTTATGGG	452
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAATGGTGGA		GATGGAgcaattcaa
		TTTCCATACACTGAA		catacagagaATGGT
		ATGATTGTTCATCTA		GGATTTCCATACACT
				GAAATGATTGTTCAT
				CTA
KATERI	CS147	TCAGCAACTTATGGG	421	TCAGCAACTTATGGG	453
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS150	TCAGCAACTTATGGG	422	TCAGCAACTTATGGG	454
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACACAGAGAATGGT		ACatACAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
KATERI	CS151	TCAGCAACTTATGGG	423	TCAGCAACTTATGGG	455
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACACAGAGAATGGT		ACatACAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
KATERI	CS148	TCAGCAACTTATGGG	424	TCAGCAACTTATGGG	456
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS149	TCAGCAACTTATGGG	425	TCAGCAACTTATGGG	457
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS152	TCAGCAACTTATGGG	426	TCAGCAACTTATGGG	458
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAGAATGGT		ACATacAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
KATERI	CS153	TCAGCAACTTATGGG	427	TCAGCAACTTATGGG	459
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		GAGAATGGTGGATTT		catacaGAGAATGGTGG
		CCATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTA		AATGATTGTTCATCT
				A
KATERI	CS102	TCAGCAACTTATGGG	428	TCAGCAACTTATGGG	460
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS103	TCAGCAACTTATGGG	429	TCAGCAACTTATGGG	461
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAGAATGGT		ACATacAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
TN90	CS143	TCAGCAACTTATGGG	430	TCAGCAACTTATGGG	462
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACAAATGGTGGATTT		ACAtacagagAATGGTG
		CCATACACTGAAATG		GATTTCCATACACTG
		ATTGTTCATCTA		AAATGATTGTTCATC
				TA
TN90	18GH	TCAGCAACTTATGGG	431	TCAGCAACTTATGGG	463
	2169	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAGAATGGT		ACATacAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
TN90	CS120	TCAGCAACTTATGGG	432	TCAGCAACTTATGGG	464
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	17GH	TCAGCAACTTATGGG	433	TCAGCAACTTATGGG	465
	1698-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	22	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	17GH	TCAGCAACTTATGGG	434	TCAGCAACTTATGGG	466
	1700-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	13	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	17GH	TCAGCAACTTATGGG	435	TCAGCAACTTATGGG	467
	1702-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	17	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	18GH	TCAGCAACTTATGGG	436	TCAGCAACTTATGGG	468
	2171	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	CS165	TCAGCAACTTATGGG	437	TCAGCAACTTATGGG	469
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	CS118	TCAGCAACTTATGGG	438	TCAGCAACTTATGGG	470
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATATACAGAGAAT		GATggagcaattcaa
		GGTGGATTTCCATAC		cATACAGAGAATGGT
		ACTGAAATGATTGTT		GGATTTCCATACACT
		CATCTA		GAAATGATTGTTCAT
				CTA
TN90	CS113	TCAGCAACTTATGGG	439	TCAGCAACTTATGGG	471
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		A		CTA
TN90	17GH	TCAGCAACTTATGGG	440	TCAGCAACTTATGGG	472
	1737-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	24	GATGGAGCAATTCA		GATGGAGCAATTCA
		ATACAGAGAATGGT		AcaTACAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
IZMIR	18GH	TCAGCAACTTATGGG	441	TCAGCAACTTATGGG	473
	2254-7	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		GAGAATGGTGGATTT		catacaGAGAATGGT
		CCATACACTGAAATG		GGATTTCCATACACT
		ATTGTTCATCTA		GAAATGATTGTTCAT
				CTA

TABLE 12B
A list of exemplary mutant alleles obtained
in the PMT1b gene. Mutant allele sequences
listed here represent approximately 90-
nucleotide-long genomic sequences from each
edited PMT1b gene with the edited site in
the middle of the genomic sequence (e.g.,
45 nucleotides on each side of the deleted
sequence site). The mutant allele corresponds
to the indel provided for each line in
Table 10. The lowercase letters in the
reference allele sequence (SEQ ID NO: 7)
denote which nucleotides are deleted in
the mutant allele.

			Mutant		Reference
			Allele	Reference	Allele
		Mutant Allele	SEQ	Allele	SEQ
Genotype	Line	Sequence	ID NO.	Sequence	ID NO.
BASMA	CS107	TCAGCAACTTATGGG	474	TCAGCAACTTATGGG	506
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
BASMA	CS106	TCAGCAACTTATGGG	475	TCAGCAACTTATGGG	507
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	CS115	TCAGCAACTTATGGG	476	TCAGCAACTTATGGG	508
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	18GH	TCAGCAACTTATGGG	477	TCAGCAACTTATGGG	509
	2162	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	CS111	TCAGCAACTTATGGG	478	TCAGCAACTTATGGG	510
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
K326	CS112	TCAGCAACTTATGGG	479	TCAGCAACTTATGGG	511
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAGAATGGT		ACATacAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
K326	17GH	TCAGCAACTTATGGG	480	TCAGCAACTTATGGG	512
	1678-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	60	GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAGAATGGT		ACATacAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
K326	CS131	TCAGCAACTTATGGG	481	TCAGCAACTTATGGG	513
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS164	TCAGCAACTTATGGG	482	TCAGCAACTTATGGG	514
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		GAGAATGGTGGATTT		catacaGAGAATGGTGG
		CCATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTA		AATGATTGTTCATCT
				A
KATERI	CS163	TCAGCAACTTATGGG	483	TCAGCAACTTATGGG	515
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACACAGAGAATGGT		ACatACAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
KATERI	CS146	TCAGCAACTTATGGG	484	TCAGCAACTTATGGG	516
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS147	TCAGCAACTTATGGG	485	TCAGCAACTTATGGG	517
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		GAGAATGGTGGATTT		catacaGAGAATGGTGG
		CCATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTA		AATGATTGTTCATCT
				A
KATERI	CS150	TCAGCAACTTATGGG	486	TCAGCAACTTATGGG	518
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS151	TCAGCAACTTATGGG	487	TCAGCAACTTATGGG	519
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS148	TCAGCAACTTATGGG	488	TCAGCAACTTATGGG	520
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS149	TCAGCAACTTATGGG	489	TCAGCAACTTATGGG	521
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS152	TCAGCAACTTATGGG	490	TCAGCAACTTATGGG	522
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
KATERI	CS153	TCAGCAACTTATGGG	491	TCAGCAACTTATGGG	523
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ATACAGAGAATGGT		AcaTACAGAGAATGG
		GGATTTCCATACACT		TGGATTTCCATACAC
		GAAATGATTGTTCAT		TGAAATGATTGTTCA
		CTA		TCTA
KATERI	CS102	TCAGCAACTTATGGG	492	TCAGCAACTTATGGG	524
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		CAGAGAATGGTGGA		cataCAGAGAATGGTG
		TTTCCATACACTGAA		GATTTCCATACACTG
		ATGATTGTTCATCTA		AAATGATTGTTCATC
				TA
KATERI	CS103	TCAGCAACTTATGGG	493	TCAGCAACTTATGGG	525
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		GAGAATGGTGGATTT		catacaGAGAATGGTGG
		CCATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTA		AATGATTGTTCATCT
				A
TN90	CS143	TCAGCAACTTATGGG	494	TCAGCAACTTATGGG	526
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	18GH	TCAGCAACTTATGGG	495	TCAGCAACTTATGGG	527
	2169	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	CS120	TCAGCAACTTATGGG	496	TCAGCAACTTATGGG	528
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAATGGTGG		ACATacagAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		A		CTA
TN90	17GH	TCAGCAACTTATGGG	497	TCAGCAACTTATGGG	529
	1698-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	22	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	17GH	TCAGCAACTTATGGG	498	TCAGCAACTTATGGG	530
	1700-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	13	GATGGAGCAATTCA		GATGGAGCAATTCAa
		CAGAGAATGGTGGA		cataCAGAGAATGGTG
		TTTCCATACACTGAA		GATTTCCATACACTG
		ATGATTGTTCATCTA		AAATGATTGTTCATC
				TA
TN90	17GH	TCAGCAACTTATGGG	499	TCAGCAACTTATGGG	531
	1702-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	17	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	18GH	TCAGCAACTTATGGG	500	TCAGCAACTTATGGG	532
	2171	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	CS165	TCAGCAACTTATGGG	501	TCAGCAACTTATGGG	533
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
TN90	CS118	TCAGCAACTTATGGG	502	TCAGCAACTTATGGG	534
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTAG		GATGGAGCAATTcaac
		AATGGTGGATTTCCA		atacagAGAATGGTGGA
		TACACTGAAATGATT		TTTCCATACACTGAA
		GTTCATCTA		ATGATTGTTCATCTA
TN90	CS113	TCAGCAACTTATGGG	503	TCAGCAACTTATGGG	535
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACATAGAATGGTGG		ACATacagAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		A		CTA
TN90	17GH	TCAGCAACTTATGGG	504	TCAGCAACTTATGGG	536
	1737-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	24	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		catACAGAGAATGGTG
		ATTTCCATACACTGA		GATTTCCATACACTG
		AATGATTGTTCATCT		AAATGATTGTTCATC
		A		TA
IZMIR	18GH	TCAGCAACTTATGGG	505	TCAGCAACTTATGGG	537
	2254-7	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCA
		ACACTGGTGGATTTC		AcatacagagACTGGTGG
		CATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTA		AATGATTGTTCATCT
				A

TABLE 12C
A list of exemplary mutant alleles obtained
in the PMT2 gene. Mutant allele sequences
listed here represent approximately
90-nucleotide-long genomic sequences from
each edited PMT2 gene with the edited site
in the middle of the genomic sequence (e.g.,
45 nucleotides on each side of the deleted
sequence site). The mutant allele corresponds
to the indel provided for each line in Table 10.
The lowercase letters in the reference allele
sequence (SEQ ID NO: 8) denote which nucleotides
are deleted in the mutant allele.

			Mutant		Reference
			Allele	Reference	Allele
		Mutant Allele	SEQ	Allele	SEQ
Genotype	Line	Sequence	ID NO.	Sequence	ID NO.
BASMA	CS107	TCAGCAACTTATGGG	538	TCAGCAACTTATGGG	570
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
BASMA	CS106	TCAGCAACTTATGGG	539	TCAGCAACTTATGGG	571
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
K326	CS115	TCAGCAACTTATGGG	540	TCAGCAACTTATGGG	572
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
K326	18GH	TCAGCAACTTATGGG	541	TCAGCAACTTATGGG	573
	2162	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		AGAATGGTGGATTTC		cacacagAGAATGGTGG
		CATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTT		AATGATTGTTCATCT
				T
K326	CS111	TCAGCAACTTATGGG	542	TCAGCAACTTATGGG	574
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
K326	CS112	TCAGCAACTTATGGG	543	TCAGCAACTTATGGG	575
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
K326	17GH	TCAGCAACTTATGGG	544	TCAGCAACTTATGGG	576
	1678-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	60	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
K326	CS131	TCAGCAACTTATGGG	545	TCAGCAACTTATGGG	577
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
KATERI	CS164	TCAGCAACTTATGGG	546	TCAGCAACTTATGGG	578
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS163	TCAGCAACTTATGGG	547	TCAGCAACTTATGGG	579
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		CACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS146	TCAGCAACTTATGGG	548	TCAGCAACTTATGGG	580
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		CACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS147	TCAGCAACTTATGGG	549	TCAGCAACTTATGGG	581
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		CACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS150	TCAGCAACTTATGGG	550	TCAGCAACTTATGGG	582
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		CACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS151	TCAGCAACTTATGGG	551	TCAGCAACTTATGGG	583
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		CACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS148	TCAGCAACTTATGGG	552	TCAGCAACTTATGGG	584
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		CACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS149	TCAGCAACTTATGGG	553	TCAGCAACTTATGGG	585
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS152	TCAGCAACTTATGGG	554	TCAGCAACTTATGGG	586
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS153	TCAGCAACTTATGGG	555	TCAGCAACTTATGGG	587
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
KATERI	CS102	TCAGCAACTTATGGG	556	TCAGCAACTTATGGG	588
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		AGAATGGTGGATTTC		cacacagAGAATGGTGG
		CATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTT		AATGATTGTTCATCT
				T
KATERI	CS103	TCAGCAACTTATGGG	557	TCAGCAACTTATGGG	589
NI		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		AGAATGGTGGATTTC		cacacagAGAATGGTGG
		CATACACTGAAATG		ATTTCCATACACTGA
		ATTGTTCATCTT		AATGATTGTTCATCT
				T
TN90	CS143	TCAGCAACTTATGGG	558	TCAGCAACTTATGGG	590
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
TN90	18GH	TCAGCAACTTATGGG	559	TCAGCAACTTATGGG	591
	2169	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
TN90	CS120	TCAGCAACTTATGGG	560	TCAGCAACTTATGGG	592
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
TN90	17GH	TCAGCAACTTATGGG	561	TCAGCAACTTATGGG	593
	1698-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	22	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
TN90	17GH	TCAGCAACTTATGGG	562	TCAGCAACTTATGGG	594
	1700-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	13	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
TN90	17GH	TCAGCAACTTATGGG	563	TCAGCAACTTATGGG	595
	1702-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	17	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACACAGAGAATGGT		cACACAGAGAATGGT
		GGATTTCCATACACT		GGATTTCCATACACT
		GAAATGATTGTTCAT		GAAATGATTGTTCAT
		CTT		CTT
TN90	18GH	TCAGCAACTTATGGG	564	TCAGCAACTTATGGG	596
	2171	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
TN90	CS165	TCAGCAACTTATGGG	565	TCAGCAACTTATGGG	597
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
TN90	CS118	TCAGCAACTTATGGG	566	TCAGCAACTTATGGG	598
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
TN90	CS113	TCAGCAACTTATGGG	567	TCAGCAACTTATGGG	599
		AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
		GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
TN90	17GH	TCAGCAACTTATGGG	568	TCAGCAACTTATGGG	600
	1737-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	24	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT
IZMIR	18GH	TCAGCAACTTATGGG	569	TCAGCAACTTATGGG	601
	2254-	AAGGTTCTGACTTTG		AAGGTTCTGACTTTG
	7	GATGGAGCAATTCA		GATGGAGCAATTCAa
		ACAGAGAATGGTGG		cacACAGAGAATGGT
		ATTTCCATACACTGA		GGATTTCCATACACT
		AATGATTGTTCATCT		GAAATGATTGTTCAT
		T		CTT

TABLE 12D
A list of exemplary mutant alleles obtained in
the PMT3 gene. Mutant allele sequences listed
here represent approximately 90-nucleotide-long
genomic sequences from each edited PMT3 gene
with the edited site in the middle of the genomic
sequence (e.g., 45 nucleotides on each side of the
deleted sequence site). The mutant allele
corresponds to the indel provided for each line in
Table 10. The lowercase letters in the reference
allele sequence (SEQ ID NO: 9) denote which
nucleotides are deleted in the mutant allele.

			Mutant		Reference
			Allele	Reference	Allele
		Mutant Allele	SEQ	Allele	SEQ
Genotype	Line	Sequence	ID NO.	Sequence	ID NO.
BASMA	CS107	TCAGCAACATATGG	602	TCAGCAACATATGG	634
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
BASMA	CS106	TCAGCAACATATGG	603	TCAGCAACATATGG	635
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
K326	CS115	TCAGCAACATATGG	604	TCAGCAACATATGG	636
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AAAGAGAATGGTGG		AACacacAGAGAATGG
		ATTTCCATACACTGA		TGGATTTCCATACAC
		AATGATTGTTCATCT		TGAAATGATTGTTCA
		T		TCTT
K326	18GH	TCAGCAACATATGG	605	TCAGCAACATATGG	637
	2162	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
K326	CS111	TCAGCAACATATGG	606	TCAGCAACATATGG	638
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
K326	CS112	TCAGCAACATATGG	607	TCAGCAACATATGG	639
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
K326	17GH	TCAGCAACATATGG	608	TCAGCAACATATGG	640
	1678-	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
	60	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
K326	CS131	TCAGCAACATATGG	609	TCAGCAACATATGG	641
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTCa
		AGAATGGTGGATTTC		acacacagAGAATGGTG
		CATACACTGAAATG		GATTTCCATACACTG
		ATTGTTCATCTT		AAATGATTGTTCATC
				TT
KATERI	CS164	TCAGCAACATATGG	610	TCAGCAACATATGG	642
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS163	TCAGCAACATATGG	611	TCAGCAACATATGG	643
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS146	TCAGCAACATATGG	612	TCAGCAACATATGG	644
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTca
		AGAGAATGGTGGAT		acacaCAGAGAATGGT
		TTCCATACACTGAAA		GGATTTCCATACACT
		TGATTGTTCATCTT		GAAATGATTGTTCAT
				CTT
KATERI	CS147	TCAGCAACATATGG	613	TCAGCAACATATGG	645
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTca
		AGAGAATGGTGGAT		acacaCAGAGAATGGT
		TTCCATACACTGAAA		GGATTTCCATACACT
		TGATTGTTCATCTT		GAAATGATTGTTCAT
				CTT
KATERI	CS150	TCAGCAACATATGG	614	TCAGCAACATATGG	646
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AAAGAATGGTGGAT		AAcacacagAGAATGGT
		TTCCATACACTGAAA		GGATTTCCATACACT
		TGATTGTTCATCTT		GAAATGATTGTTCAT
				CTT
KATERI	CS151	TCAGCAACATATGG	615	TCAGCAACATATGG	647
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS148	TCAGCAACATATGG	616	TCAGCAACATATGG	648
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AAAGAATGGTGGAT		AAcacacagAGAATGGT
		TTCCATACACTGAAA		GGATTTCCATACACT
		TGATTGTTCATCTT		GAAATGATTGTTCAT
				CTT
KATERI	CS149	TCAGCAACATATGG	617	TCAGCAACATATGG	649
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS152	TCAGCAACATATGG	618	TCAGCAACATATGG	650
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS153	TCAGCAACATATGG	619	TCAGCAACATATGG	651
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS102	TCAGCAACATATGG	620	TCAGCAACATATGG	652
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS103	TCAGCAACATATGG	621	TCAGCAACATATGG	653
NI		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	CS143	TCAGCAACATATGG	622	TCAGCAACATATGG	654
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	18GH	TCAGCAACATATGG	623	TCAGCAACATATGG	655
	2169	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
TN90	CS120	TCAGCAACATATGG	624	TCAGCAACATATGG	656
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	17GH	TCAGCAACATATGG	625	TCAGCAACATATGG	657
	1698-	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
	22	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAATGGTG		AACACacagAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
TN90	17GH	TCAGCAACATATGG	626	TCAGCAACATATGG	658
	1700-	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
	13	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	17GH	TCAGCAACATATGG	627	TCAGCAACATATGG	659
	1702-	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
	17	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	18GH	TCAGCAACATATGG	628	TCAGCAACATATGG	660
	2171	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTca
		AGAGAATGGTGGAT		acacaCAGAGAATGGT
		TTCCATACACTGAAA		GGATTTCCATACACT
		TGATTGTTCATCTT		GAAATGATTGTTCAT
				CTT
TN90	CS165	TCAGCAACATATGG	629	TCAGCAACATATGG	661
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
TN90	CS118	TCAGCAACATATGG	630	TCAGCAACATATGG	662
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	CS113	TCAGCAACATATGG	631	TCAGCAACATATGG	663
		GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	17GH	TCAGCAACATATGG	632	TCAGCAACATATGG	664
	1737-	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
	24	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
IZMIR	18GH	TCAGCAACATATGG	633	TCAGCAACATATGG	665
	2254-7	GAAGGTTCTGACTTT		GAAGGTTCTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT

TABLE 12E
A list of exemplary mutant alleles obtained
in the PMT4 gene. Mutant allele sequences
listed here represent approximately
90-nucleotide-long genomic sequences from
each edited PMT4 gene with the edited site
in the middle of the genomic sequence (e.g.,
45 nucleotides on each side of the deleted
sequence site). The mutant allele corresponds
to the indel provided for each line in Table 10.
The lowercase letters in the reference allele
sequence (SEQ ID NO: 10) denote which nucleotides
are deleted in the mutant allele.

			Mutant		Reference
			Allele	Reference	Allele
		Mutant Allele	SEQ	Allele	SEQ
Genotype	Line	Sequence	ID NO.	Sequence	ID NO.
BASMA	CS107	TCAGCAACATATGG	666	TCAGCAACATATGG	698
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
BASMA	CS106	TCAGCAACATATGG	667	TCAGCAACATATGG	699
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
K326	CS115	TCAGCAACATATGG	668	TCAGCAACATATGG	700
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACGAGAATGGTGG		AACacacaGAGAATGG
		ATTTCCATACACTGA		TGGATTTCCATACAC
		AATGATTGTTCATCT		TGAAATGATTGTTCA
		T		TCTT
K326	18GH	TCAGCAACATATGG	669	TCAGCAACATATGG	701
	2162	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACGAGAATGGTGG		AACacacaGAGAATGG
		ATTTCCATACACTGA		TGGATTTCCATACAC
		AATGATTGTTCATCT		TGAAATGATTGTTCA
		T		TCTT
K326	CS111	TCAGCAACATATGG	670	TCAGCAACATATGG	702
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AAAGAGAATGGTGG		AAcacacAGAGAATGG
		ATTTCCATACACTGA		TGGATTTCCATACAC
		AATGATTGTTCATCT		TGAAATGATTGTTCA
		T		TCTT
K326	CS112	TCAGCAACATATGG	671	TCAGCAACATATGG	703
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AAAGAGAATGGTGG		AAcacacAGAGAATGG
		ATTTCCATACACTGA		TGGATTTCCATACAC
		AATGATTGTTCATCT		TGAAATGATTGTTCA
		T		TCTT
K326	17GH	TCAGCAACATATGG	672	TCAGCAACATATGG	704
	1678-	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
	60	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
K326	CS131	TCAGCAACATATGG	673	TCAGCAACATATGG	705
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS164	TCAGCAACATATGG	674	TCAGCAACATATGG	706
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS163	TCAGCAACATATGG	675	TCAGCAACATATGG	707
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS146	TCAGCAACATATGG	676	TCAGCAACATATGG	708
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS147	TCAGCAACATATGG	677	TCAGCAACATATGG	709
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS150	TCAGCAACATATGG	678	TCAGCAACATATGG	710
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS151	TCAGCAACATATGG	679	TCAGCAACATATGG	711
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTA		GGATGGAGCAATTca
		GAATGGTGGATTTCC		acacacagAGAATGGTG
		ATACACTGAAATGAT		GATTTCCATACACTG
		TGTTCATCTT		AAATGATTGTTCATC
				TT
KATERI	CS148	TCAGCAACATATGG	680	TCAGCAACATATGG	712
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS149	TCAGCAACATATGG	681	TCAGCAACATATGG	713
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTA		GGATGGAGCAATTca
		GAATGGTGGATTTCC		acacacagAGAATGGTG
		ATACACTGAAATGAT		GATTTCCATACACTG
		TGTTCATCTT		AAATGATTGTTCATC
				TT
KATERI	CS152	TCAGCAACATATGG	682	TCAGCAACATATGG	714
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS153	TCAGCAACATATGG	683	TCAGCAACATATGG	715
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
KATERI	CS102	TCAGCAACATATGG	684	TCAGCAACATATGG	716
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
KATERI	CS103	TCAGCAACATATGG	685	TCAGCAACATATGG	717
NI		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	CS143	TCAGCAACATATGG	686	TCAGCAACATATGG	718
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
TN90	18GH	TCAGCAACATATGG	687	TCAGCAACATATGG	719
	2169	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	CS120	TCAGCAACATATGG	688	TCAGCAACATATGG	720
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	17GH	TCAGCAACATATGG	689	TCAGCAACATATGG	721
	1698-	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
	22	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	17GH	TCAGCAACATATGG	690	TCAGCAACATATGG	722
	1700-	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
	13	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
TN90	17GH	TCAGCAACATATGG	691	TCAGCAACATATGG	723
	1702-	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
	17	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAAGAATGGTGG		AACAcacagAGAATGG
		ATTTCCATACACTGA		TGGATTTCCATACAC
		AATGATTGTTCATCT		TGAAATGATTGTTCA
		T		TCTT
TN90	18GH	TCAGCAACATATGG	692	TCAGCAACATATGG	724
	2171	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	CS165	TCAGCAACATATGG	693	TCAGCAACATATGG	725
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACAGAGAATGGTG		AacacACAGAGAATGG
		GATTTCCATACACTG		TGGATTTCCATACAC
		AAATGATTGTTCATC		TGAAATGATTGTTCA
		TT		TCTT
TN90	CS118	TCAGCAACATATGG	694	TCAGCAACATATGG	726
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	CS113	TCAGCAACATATGG	695	TCAGCAACATATGG	727
		GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
TN90	17GH	TCAGCAACATATGG	696	TCAGCAACATATGG	728
	1737-	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
	24	GGATGGAGCAATTC		GGATGGAGCAATTC
		AACACAGAGAATGG		AacACACAGAGAATG
		TGGATTTCCATACAC		GTGGATTTCCATACA
		TGAAATGATTGTTCA		CTGAAATGATTGTTC
		TCTT		ATCTT
IZMIR	18GH	TCAGCAACATATGG	697	TCAGCAACATATGG	729
	2254-7	GAAGGTTTTGACTTT		GAAGGTTTTGACTTT
		GGATGGAGCAATTC		GGATGGAGCAATTC
		AAGAATGGTGGATTT		AacacacagAGAATGGT
		CCATACACTGAAATG		GGATTTCCATACACT
		ATTGTTCATCTT		GAAATGATTGTTCAT
				CTT

Example 8. Alkaloid Analysis of PMT Edited Lines

Homozygous genome edited tobacco lines from Example 7, along with control lines, are grown in in a field. At flowering stage, plants are topped and two-weeks post topping, lamina samples are collected from the third, fourth, and fifth leaves from the top of the plant and alkaloid levels are measured (see Tables 13A-13C) using a method in accordance with CORESTA Method No 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and those defined in the Centers for Disease Control and Prevention's Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, as published in the Federal Register Vol. 64, No. 55 Mar. 23, 1999 (and as amended in Vol. 74, No. 4, Jan. 7, 2009).

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 %) on either on as is or dry weight basis. Reporting data on a dry weight basis requires an oven volatiles (OV) determination. Unless specified otherwise, total or individual alkaloid levels or nicotine levels shown herein are on a dry weight basis (e.g., percent total alkaloid or percent nicotine).

Plants are also planted in the field, harvested, and tested for alkaloids and TSNA levels in cured tobacco. Both leaf yield and leaf grade are also assessed for PMT edited plants.

TABLE 13A
Nicotine analysis of K326 and TN90 PMT edited
lines after two weeks after flowering.

				Nicotine
	Variety	Line	Replicate	(mg/g tissue)

	K 326	CS111	1	0.023
			2	0.024
		CS131	1	0.022
			2	0.018
			3	0.021
		CS115	1	0.023
			2	0.015
		Control	1	16.8
			2	17.2
			3	16.6
	TN 90 LC	CS116	1	0.029
			2	0.022
		CS133	1	0.016
			2	0.018
		CS135	1	0.027
			2	0.031
		CS120	1	0.022
			2	0.045
		CS137	1	0.07
			2	0.048
		Control	1	29.5
			2	29.8
			3	24.2

TABLE 13B
Nicotine analysis of K326 and TN90 PMT edited
lines after two-weeks after topping.

				Nicotine
	Variety	Group	Replicate	(mg/g tissue)

	K 326	CS111	1	0.024
			2	0.025
		CS131	1	0.021
			2	0.02
			3	0.019
		CS115	1	0.018
		Control	1	16.771
			2	17.212
			3	16.581
			4	22.734
	TN 90 LC	CS116	1	0.015
		CS133	1	0.036
			2	0.018
		CS135	1	0.018
			2	0.019
		CS120	1	0.04
			2	0.025
		CS137	1	0.051
			2	0.057
		Control	1	29.472
			2	29.776
			3	24.22
			4	24.939

TABLE 13C
Nicotine analysis of Katerini and Basma PMT
edited lines after two-weeks after flowering.

				Nicotine
	Variety	Group	Replicate	(mg/g tissue)

	Katerini	CS102	1	0.032
			2	0.028
		CS103	1	0.017
		Control	1	26.109
			2	26.466
			3	27.091
	Basma	CS107	1	0.029
		CS108	1	0.014
			2	0.018
		Control	1	21.979
			2	20.88
			3	23.499

Example 9. Development of Male Sterile PMT Edited Lines

PMT edited hybrid lines are developed using the lines from Example 7. Hybrid lines are grown in the field and used as progenitors for male sterile lines. See Table 14.

TABLE 14
PMT edited very low nicotine male sterile lines

	Male Sterile	Pollen source	F1 hybrid seed
	Variety	(line)	(line)
	MS Katerini	CS102	dCS11
		CS103	dCS12
	MS Basma	CS106	dCS13
		CS107	dCS14
	MS K326	CS111	dCS15
		CS115	dCS16
	MS TN90	CS118	dCS17
		CS120	dCS18
	MS Izmir	18GH2254	dS2697

Example 10. PMT Edited Lines Resist Mold During Curing

Tobacco leaf harvested from several low alkaloid tobacco lines is subjected to standard air curing practices. The tobacco leaves are examined for mold after the completion of curing.

Tobacco from the LA BU 21 exhibits more mold infestation than TN90 LC, a TN90 variety comprising an RNAi construct to downregulate all five PMT genes, a TN90 variety comprising an RNAi construct to downregulate the alkaloid biosynthesis gene PR50, and four PMT edited lines (CS47, CS59, CS63, and CS64) in a TN90 genetic background. See Table 15 and FIGS. 6A to 6E and 7.

TABLE 15
Mold damage exhibited by tobacco lines.

Mold Rating

Percentage of Mold

	Replicate	Replicate	Replicate	Replicate	Significant	Some	Little/No
Variety	1	2	3	4	Mold	Mold	Mold

TN90 LC

G

G

G

G

G

G

G

G

G

G

G

G

0%

0%

100%

LA BU 21

G

S

G

B

S

G

S

S

S

B

S

S

17%

58%

25%

TN90

G

G

G

G

G

G

G

G

G

G

G

G

0%

0%

100%

(PMT

RNAi)

TN90

G

G

G

G

G

G

G

G

G

G

G

G

0%

0%

100%

(PR50

RNai)

CS47

G

G

G

G

G

G

G

G

G

G

G

G

0%

0%

100%

CS59

G

G

G

G

G

G

G

G

G

G

G

G

0%

0%

100%

CS63

G

G

G

G

G

G

G

G

G

G

G

G

0%

0%

100%

CS64

G

G

G

G

G

G

G

G

G

S

S

G

0%

17%

83%

“G” refers to little/no mold; “S” refers to some mold; and “B” refers to significant mold. Percentage of Mold refers to the percentage of air cured sticks of tobacco exhibited each category of mold damage.