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

Information

  • Patent Grant
  • 11384361
  • Patent Number
    11,384,361
  • Date Filed
    Friday, July 26, 2019
    5 years ago
  • Date Issued
    Tuesday, July 12, 2022
    2 years ago
Abstract
The present disclosure provides compositions and methods related to 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.
Description
INCORPORATION OF SEQUENCE LISTING

A sequence listing contained in the file named “P34620US02_SL.txt” which is 200,704 bytes (measured in MS-Windows®) and created on Jul. 24, 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 pint mutant, a double pint mutant, a triple mutant, a quadruple mutant, or a quintuple mutant. In an aspect, the present disclosure provides a tobacco plant comprising a pint 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, respectively, from a TN90 reference genome.


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


SEQ ID Nos: 11 to 15 set forth exemplary polypeptide sequences of PMT1b, PMT1a, PMT2, PMT3, and PMT4, respectively, 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 “pint 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 quintuple pmt mutant tobacco plant is provided. In another aspect, a quintuple 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 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 pint 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 pint 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 pint mutant is homozygous or heteroallelic in all five PMT genes. In another aspect, a pint 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 a pmt 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 pint 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 aldehyde 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, N.Y. (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 pint 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 pint 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 pint 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 pint 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 pint 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 pint 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 pint 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 pint 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 further aspect, a pint 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 pint 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 pint 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 pint 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-l11, Speight G-l17, 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 NC 196.


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, Md., 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, Va. 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, Md., dark fire-cured, or Oriental type are listed only for exemplary purposes. Any additional dark air-cured, Burley, Md., 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 3.5% (dry weight) of its parent, Burley 21. LA B U 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 pint 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 pint 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, Penicillium, 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
cDNA
Protein



(including regions such as
Sequence
Sequence



promoter, 5′ UTR, introns, 3′ UTR,
(SEQ ID
(SEQ ID


Gene Name
and terminator) (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







PMT1a 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
Technology

Target genes














No.
(GET)
gRNA sequence
PMT1b
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. 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.













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

















TABLE 4A





Mutant pmt 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

















Deleted

Deleted

Deleted


VARIETY
LINE
Position
sequence
Position
sequence
Position
sequence





K326
17GH1678
416..419
ATAC
415..421
CATACAG
348..349
AC




418..419
AC
417..420
TACA







K326
17GH1680
414..417
ACAT
414..417
ACAT
348..349
AC






416..417
AT







K326
17GH1804
414..417
ACAT
411..420
TCAACATACA(379)
348..349
AC






417..420
TACA







K326
17GH1898
414..417
ACAT
411..420
TCAACATACA(379)
348..349
AC






417..420
TACA







K326
18GH207
414..417
ACAT
415..415
C
348..351
ACAC






417..417
T







K326
18GH343
414..417
ACAT
414..417
ACAT
348..349
AC






416..417
AT







K326
18GH348
414..417
ACAT
414..417
ACAT
348..349
AC





K326
18GH349
414..417
ACAT
414..417
ACAT
348..349
AC





K326
18GH355
414..417
ACAT
414..417
ACAT
348..351
ACAC






416..417
AT
350..351
AC





K326
18GH359
414..417
ACAT
411..420
TCAACATACA(379)
348..349
AC






417..420
TACA







K326
18GH64
413..420
AACATACA
414..417
ACAT
349..352
CACA




417..420
TACA


354..359
AGAGAA





K326
18GH682
415..421
CATACAG
413..422
AACATACAGA(386)
348..351
ACAC






417..420
TACA
350..351
AC





K326
18GH692
414..417
ACAT
414..420
ACATACA
348..351
ACAC






416..420
ATACA
350..351
AC





K326
18GH697
414..417
ACAT
414..417
ACAT
348..351
ACAC





K326
18GH922
414..417
ACAT
414..417
ACAT
348..351
ACAC





K326
18GH957
414..417
ACAT
414..417
ACAT
349..355
CACACAG








351..354
CACA





K326
17GH1886




346..350
CAACA








349..350
CA





K326
17GH1888


418..419
AC
348..349
AC





K326
17GH1889


418..419
AC
348..349
AC





K326
17GH1892
413..419
AACATAC
414..417
ACAT
348..351
ACAC




414..419
ACATAC


350..351
AC




416..419
ATAC









K326
17GH1893
416..421
ATACAG
416..420
ATACA
348..349
AC





K326
17GH1901
414..417
ACAT
417..420
TACA
348..355
ACACACAG





K326
17GH1902
414..417
ACAT
417..420
TACA
348..355
ACACACAG





K326
17GH1808
414..417
ACAT
413..421
AACATACAG
352..354
ACA






418..421
ACAG







K326
17GH1810
414..417
ACAT
417..420
TACA
348..355
ACACACAG





K326
18GH3


414..417
ACAT
348..349
AC





K326
18GH4


414..417
ACAT
348..349
AC
















PMT3
PMT4



















Deleted

Deleted




VARIETY
LINE
Position
sequence
Position
sequence







K326
17GH1678
432..435
ACAC
547..551
CACAC








548..551
ACAC







K326
17GH1680
432..435
ACAC
546..547
AC







K326
17GH1804
433..437
CACAC
548..552
ACACA







K326
17GH1898
433..437
CACAC
548..552
ACACA







K326
18GH207
432..433
AC
546..549
ACAC







K326
18GH343
432..435
ACAC
546..547
AC







K326
18GH348
429..439
TCAACACACAG(396)
546..547
AC







K326
18GH349
429..439
TCAACACACAG(396)
546..547
AC







K326
18GH355
431..438
AACACACA
546..547
AC






435..438
CACA
550..553
ACAG






440..442
AGA









K326
18GH359
433..437
CACAC
548..552
ACACA







K326
18GH64
432..433
AC
546..549
ACAC







K326
18GH682
432..439
ACACACAG
543..554
TCAACA









CACAGA(404)






437..438
CA
549..552
CACA







K326
18GH692
432..439
ACACACAG
546..549
ACAC






437..438
CA









K326
18GH697
430..436
CAACACA
546..549
ACAC







K326
18GH922
430..436
CAACACA
546..549
ACAC







K326
18GH957
431..438
AACACACA
546..547
AC







K326
17GH1886
432..433
AC









K326
17GH1888











K326
17GH1889











K326
17GH1892
432..435
ACAC








434..435
AC









K326
17GH1893
430..436
CAACACA
546..547
AC







K326
17GH1901
432..435
ACAC
548..552
ACACA






434..435
AC
550..552
ACA







K326
17GH1902
432..435
ACAC
548..552
ACACA






434..435
AC
550..552
ACA







K326
17GH1808
432..435
ACAC
546..547
AC






434..435
AC









K326
17GH1810
432..435
ACAC
548..552
ACACA






434..435
AC
550..552
ACA







K326
18GH3
432..435
ACAC









K326
18GH4
429..439
TCAACACACAG(396)
546..547
AC
















TABLE 4B





Mutant pmt alleles in TN90 produced by genome editing using GET2.





















PMT1b
PMT1a
PMT2

















Deleted

Deleted

Deleted


VARIETY
LINE
Position
sequence
Position
sequence
Position
sequence





TN90
17GH1696
414..417
ACAT
414..417
ACAT
348..349
AC





TN90
17GH1717
414..417
ACAT
414..417
ACAT
346..352
CAACACA






415..416
CA
349..352
CACA





TN90
17GH1719
414..417
ACAT
417..420
TACA
348..349
AC






417..423
TACAGAG







TN90
17GH1729
412..421
CAACATACAG
412..418
CAACATA
348..351
ACAC





(380)








414..420
ACATACA









TN90
17GH1736
418..421
ACAG
414..417
ACAT
347..357
AACACACAGAG









(391)








351..354
CACA





TN90
17GH1737
414..417
ACAT
414..417
ACAT
346..352
CAACACA






415..416
CA
349..352
CACA





TN90
17GH1739
414..417
ACAT
414..417
ACAT
348..349
AC





TN90
17GH1740
414..417
ACAT
416..419
ATAC
348..351
ACAC






418..419
AC







TN90
17GH1835
413..421
AACATACAG
417..420
TACA
350..354
ACACA




417..420
TACA
418..421
ACAG
351..362
CACAGAGAATGG









(394)





TN90
17GH1848
414..417
ACAT
417..420
TACA
348..349
AC






417..423
TACAGAG







TN90
17GH1849
414..417
ACAT
414..417
ACAT
348..351
ACAC





TN90
17GH1912
414..417
ACAT
417..420
TACA
348..349
AC






417..423
TACAGAG







TN90
17GH1937
416..419
ATAC
412..421
CAACATACAG
348..351
ACAC







(380)








417..420
TACA







TN90
17GH1940
414..417
ACAT
417..420
TACA
348..349
AC






417..423
TACAGAG







TN90
17GH1943
414..417
ACAT
414..417
ACAT
348..349
AC





TN90
17GH1944
414..417
ACAT
414..417
ACAT
348..349
AC





TN90
18GH1051
414..418
ACATA
412..418
CAACATA
348..351
ACAC




415..421
CATACAG
415..418
CATA
350..351
AC





TN90
18GH22
416..419
ATAC
412..421
CAACATACAG
348..351
ACAC







(380)








417..420
TACA







TN90
18GH34
414..417
ACAT
414..417
ACAT
348..349
AC





TN90
18GH473
414..417
ACAT
414..417
ACAT
348..351
ACAC





TN90
18GH49
412..421
CAACATACAG 
412..418
CAACATA
348..351
ACAC





(380)









TN90
18GH50
412..421
CAACATACAG 
412..418
CAACATA
348..351
ACAC





(380)








414..420
ACATACA









TN90
18GH848
414..418
ACATA
412..418
CAACATA
348..351
ACAC




415..421
CATACAG
415..418
CATA
350..351
AC





TN90
18GH850
414..417
ACAT
416..422
ATACAGA
348..349
AC






417..420
TACA







TN90
18GH851
414..417
ACAT
416..422
ATACAGA
348..349
AC






417..420
TACA







TN90
17GH1699
419..420
CA
413..420
AACATACA
348..351
ACAC




418..423
ACAGAG
419..420
CA
349..349
C




427..427
G









TN90
17GH1708
414..417
ACAT
414..415
AC
346..355
CAACACACAG(390)






418..424
ACAGAGAA








419..420
CA







TN90
17GH1722
415..420
CATACA
414..417
ACAT
348..351
ACAC




418..421
ACAG


350..351
AC





TN90
17GH1724
416..421
ATACAG
414..417
ACAT
348..351
ACAC




417..420
TACA









TN90
17GH1725
416..421
ATACAG
414..417
ACAT
348..351
ACAC




417..420
TACA









TN90
17GH1845
416..418
ATA
412..418
CAACATA
348..351
ACAC




418..419
AC
415..418
CATA







TN90
17GH1846
415..420
CATACA
414..417
ACAT
348..351
ACAC




418..421
ACAG


350..351
AC





TN90
17GH1847
415..420
CATACA
414..417
ACAT
348..351
ACAC




418..421
ACAG


350..351
AC





TN90
17GH1911
414..417
ACAT
417..420
TACA
348..349
AC






417..423
TACAGAG







TN90
17GH1912
414..417
ACAT
417..420
TACA
348..349
AC






417..423
TACAGAG







TN90
17GH1915


414..417
ACAT







TN90
17GH1918
414..419
ACATAC
417..420
TACA
353..361
CAGAGAATG




416..419
ATAC
418..421
ACAG







TN90
17GH1928
412..418
CAACATA
414..417
ACAT






415..418
CATA
419..421
CAG







TN90
17GH1932
414..419
ACATAC
416..419
ATAC






416..419
ATAC
418..419
AC







TN90
17GH1933
414..419
ACATAC
416..419
ATAC
350..355
ACACAG




416..419
ATAC
418..419
AC
351..354
CACA





TN90
17GH1936
413..421
AACATACAG
416..419
ATAC
348..351
ACAC




417..420
TACA
418..419
AC
350..351
AC





TN90
18GH20


414..419
ACATAC
348..349
AC








352..355
ACAG





TN90
18GH47
414..419
ACATAC
416..419
ATAC
348..349
AC






418..419
AC








424..425
AA







TN90
18GH28
414..419
ACATAC
414..417
ACAT
348..351
ACAC




416..419
ATAC


350..351
AC





TN90
18GH31
414..419
ACATAC
416..419
ATAC
348..349
AC






418..419
AC








424..425
AA

















PMT3
PMT4

















Deleted

Deleted



VARIETY
LINE
Position
sequence
Position
sequence






TN90
17GH1696
436..439
ACAG
546..547
AC






TN90
17GH1717
432..435
ACAC
546..547
AC





434..435
AC








TN90
17GH1719
432..433
AC
546..549
ACAC






TN90
17GH1729
432..433
AC
546..547
AC






TN90
17GH1736
432..433
AC
546..547
AC






TN90
17GH1737
432..435
ACAC
546..547
AC





434..435
AC








TN90
17GH1739
432..433
AC
546..549
ACAC







548..549
AC






TN90
17GH1740
435..438
CACA
546..547
AC





436..439
ACAG








TN90
17GH1835
432..435
ACAC
546..547
AC






TN90
17GH1848
432..433
AC
546..549
ACAC






TN90
17GH1849
430..436
CAACACA
546..549
ACAC





433..436
CACA
548..549
AC






TN90
17GH1912
432..433
AC
546..549
ACAC






TN90
17GH1937
432..435
ACAC
546..547
AC





434..435
AC








TN90
17GH1940
432..433
AC
546..549
ACAC






TN90
17GH1943
432..433
AC
546..549
ACAC






TN90
17GH1944
432..433
AC
546..549
ACAC







548..549
AC






TN90
18GH1051
432..433
AC
546..549
ACAC







548..549
AC






TN90
18GH22
432..435
ACAC
546..547
AC





434..435
AC








TN90
18GH34
432..433
AC
546..549
ACAC







548..549
AC






TN90
18GH473
430..436
CAACACA
546..549
ACAC





433..436
CACA
548..549
AC






TN90
18GH49
432..433
AC
546..547
AC






TN90
18GH50
432..433
AC
546..547
AC






TN90
18GH848
432..433
AC
546..549
ACAC






TN90
18GH850
435..438
CACA
546..547
AC





436..439
ACAG
550..553
ACAG






TN90
18GH851
435..438
CACA
546..547
AC





436..439
ACAG
550..553
ACAG






TN90
17GH1699
429..438
TCAACACACA
546..547
AC






(395)







432..446
ACACACAG








AGAATGG








(399)








TN90
17GH1708
432..437
ACACAC







440..443
AGAA








TN90
17GH1722
432..433
AC
546..549
ACAC





435..439
CACAG








TN90
17GH1724
432..435
ACAC
550..553
ACAG





434..435
AC








TN90
17GH1725
432..435
ACAC
550..553
ACAG





434..435
AC








TN90
17GH1845
433..437
CACAC
546..551
ACACAC





436..437
AC
550..551
AC






TN90
17GH1846
432..433
AC
546..549
ACAC





435..439
CACAG








TN90
17GH1847
432..433
AC
546..549
ACAC





435..439
CACAG








TN90
17GH1911
432..433
AC
546..549
ACAC






TN90
17GH1912
432..433
AC
546..549
ACAC






TN90
17GH1915
432..435
ACAC








TN90
17GH1918
432..435
ACAC
544..550
CAACACA







554..554
A







558..563
TGGTGG







565..566
TT







569..572
CATA






TN90
17GH1928
432..448
ACACACAG
546..547
AC






AGAATGGTG








(400)







437..438
CA








TN90
17GH1932


546..551
ACACAC






TN90
17GH1933
413..414
CT
546..551
ACACAC





418..419
GA







426..427
AA







431..432
AA







432..435
ACAC








TN90
17GH1936
432..433
AC
544..550
CAACACA







547..550
CACA






TN90
18GH20
432..433
AC
546..549
ACAC







548..549
AC






TN90
18GH47
436..439
ACAG
546..549
ACAC






TN90
18GH28
433..437
CACAC
546..549
ACAC







548..549
AC






TN90
18GH31
436..439
ACAG
546..549
ACAC
















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

















Deleted

Deleted

Deleted


VARIETY
LINE
Position
sequence
Position
sequence
Position
sequence





NLMz
18GH10
414..417
ACAT
414..417
ACAT
350..351
AC





NLMz
18GH1004
414..417
ACAT
412..418
CAACATA
348..349
AC




416..416
A









NLMz
18GH1033
414..417
ACAT
412..418
CAACATA
348..349
AC




416..416
A









NLMz
18GH132
417..418
TA
416..419
ATAC
348..352
ACACA






418..419
AC
348..353
ACACAC





NLMz
18GH134
414..417
ACAT
414..423
ACATACAGAG
348..351
ACAC







(388)








419..420
CA







NLMz
18GH217
414..417
ACAT
415..419
CATAC
346..352
CAACACA






417..418
TA
351..352
CA





NLMz
18GH456
416..419
ATAC
414..417
ACAT
348..349
AC




418..419
AC









NLMz
18GH457
414..417
ACAT
414..417
ACAT
348..349
AC





NLMz
18GH460
414..417
ACAT
409..420
ATTCAACA
350..363
ACACAGA







TACA(383)

GAATGGT(393)






416..429
ATACAGAG
351..354
CACA







AATGGT
353..354
CA







(389)







NLMz
18GH465
414..417
ACAT
412..418
CAACATA
348..349
AC




416..416
A









NLMz
18GH830
414..417
ACAT
414..417
ACAT
348..351
ACAC





NLMz
18GH831
413..428
AACATACAGA
413..428
AACATACA
348..351
ACAC





GAATGG(381)

GAGAATGG









(381)








417..420
TACA







NLMz
18GH836
414..417
ACAT
414..417
ACAT
348..349
AC





NLMz
18GH841
415..421
CATACAG
413..420
AACATACA
347..357
AACACAC




419..420
CA
414..420
ACATACA

AGAG(391)





NLMz
18GH974
411..420
TCAACATACA(379)
411..420
TCAACATACA
351..354
CACA







(379)






417..420
TACA


353..354
CA





NLMz
18GH981
412..418
CAACATA
414..417
ACAT
346..352
CAACACA








349..352
CACA





NLMz
18GH994
414..417
ACAT
412..418
CAACATA
348..349
AC




416..416
A









NLMz
18GH128


414..420
ACATACA
348..351
ACAC






417..421
TACAG
350..351
AC





NLMz
18GH130
414..437
ACATACAGAG
412..418
CAACATA
349..355
CACACAG





AATGGTGGAT









TTCC (382)








417..420
TACA
415..418
CATA







NLMz
18GH131
417..418
TA
416..419
ATAC
348..353
ACACAC






417..419
TAC
350..353
ACAC






418..419
AC







NLMz
18GH133
413..419
AACATAC
414..420
ACATACA
348..351
ACAC




414..419
ACATAC
417..420
TACA
350..351
AC






417..421
TACAG







NLMz
18GH136




348..349
AC





NLMz
18GH216
412..418
CAACATA
414..419
ACATAC
347..354
AACACACA




415..418
CATA
416..419
ATAC







NLMz
18GH227
418..419
AC
414..417
ACAT
348..351
ACAC





NLMz
18GH5
414..417
ACAT
414..420
ACATACA
352..355
ACAG






415..421
CATACAG







NLMz
18GH6
414..417
ACAT
416..429
ATACAGAG
350..363
ACACAGA







AATGGT(389)

GAATGGT(393)






417..422
TACAGA
351..356
CACAGA








353..356
CAGA





NLMz
18GH65
416.423
ATACAGAG
414..419
ACATAC
348..351
ACAC




418.420
ACA
417..419
TAC
350..351
AC





NLMz
18GH66
414.417
ACAT
414..420
ACATACA
352..355
ACAG






415.421
CATACAG







NLMz
18GH69


411..420
TCAACATACA









(379)







NLMz
18GH72
416..423
ATACAGAG
414..419
ACATAC
348..351
ACAC




418..420
ACA
417..419
TAC
350..351
AC





NLMz
18GH73


414..420
ACATACA
348..351
ACAC






417..421
TACAG
350..351
AC





NLMz
18GH74


412..418
CAACATA







NLMz
18GH78
414..419
ACATAC
414..417
ACAT
348..349
AC





NLMz
18GH79


414..420
ACATACA
348..351
ACAC






417..421
TACAG
350..351
AC





NLMz
18GH8
417..420
TACA
416..421
ATACAG
348..354
ACACACA






417..420
TACA
350..354
ACACA





NLMz
18GH9
417..418
TA
416..419
ATAC
348..353
ACACAC






418..419
AC







NLMz
18GH71
414..417
ACAT
414..417
ACAT
348..351
ACAC





NLMz
17GH1905
414..417
ACAT
414..420
ACATACA
352..355
ACAG






415..421
CATACAG

















PMT3
PMT4

















Deleted

Deleted



VARIETY
LINE
Position
sequence
Position
sequence






NLMz
18GH10
431..441
AACACACAGAG
546..549
ACAC






(391)








NLMz
18GH1004
430..436
CAACACA
546..553
ACACACAG





435..436
CA
551..552
CA






NLMz
18GH1033
430..436
CAACACA
546..553
ACACACAG





435..436
CA
551..552
CA






NLMz
18GH132
432..437
ACACAC
546..547
AC





434..437
ACAC







436..437
AC








NLMz
18GH134
433..439
CACACAG
550..556
ACAGAGA






NLMz
18GH217
432..435
ACAC
545..557
AACACACA








GAGAA(407)







551..552
CA






NLMz
18GH456
432..433
AC
546..549
ACAC






NLMz
18GH457
432..433
AC
546..549
ACAC






NLMz
18GH460
436..439
ACAG
550..553
ACAG






NLMz
18GH465
430..436
CAACACA
546..553
ACACACAG





435..436
CA
551..552
CA






NLMz
18GH830
432..435
ACAC
550..553
ACAG






NLMz
18GH831


546..547
AC






NLMz
18GH836
432..433
AC
546..549
ACAC






NLMz
18GH841


546..547
AC






NLMz
18GH974
432..433
AC
546..549
ACAC





436..439
ACAG








NLMz
18GH981
429..439
TCAACACA
546..552
ACACACA






CAG(396)







431..441
AACACACA
546..553
ACACACAG






GAG(391)








NLMz
18GH994
430..436
CAACACA
546..553
ACACACAG





435..436
CA
551..552
CA






NLMz
18GH128


546..547
AC






NLMz
18GH130
430..436
CAACACA
546..549
ACAC






NLMz
18GH131


546..547
AC






NLMz
18GH133


546..547
AC






NLMz
18GH136










NLMz
18GH216
432..433
AC
546..549
ACAC







548..549
AC






NLMz
18GH227


546..549
ACAC






NLMz
18GH5
429..435
TCAACAC
546..551
ACACAC





432..435
ACAC
548..551
ACAC






NLMz
18GH6
436..439
ACAG
550..553
ACAG






NLMz
18GH65
433..437
CACAC
546..549
ACAC





436..437
AC








NLMz
18GH66
429..435
TCAACAC
546..551
ACACAC





432..435
ACAC
548..551
ACAC






NLMz
18GH69
432..433
AC
546..549
ACAC





436..439
ACAG








NLMz
18GH72
433..437
CACAC
546..549
ACAC





436..437
AC








NLMz
18GH73


546..547
AC






NLMz
18GH74










NLMz
18GH78
431..431
A
546..549
ACAC





434..438
ACACA
548..549
AC





435..438
CACA








NLMz
18GH79


546..547
AC






NLMz
18GH8
435..447
CACAGAGA
549..552
CACA






ATGGT(401)
549..553
CACAG






NLMz
18GH9


546..547
AC






NLMz
18GH71
431..441
AACACACA
546..549
ACAC






GAG(391)








NLMz
17GH1905
429..435
TCAACAC
546..551
ACACAC





432..435
ACAC
548..551
ACAC
















TABLE 4D





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





















PMT1b
PMT1a
PMT2

















Deleted

Deleted

Deleted


VARIETY
LINE
Position
sequence
Position
sequence
Position
sequence





Katerini
18GH125
412..418
CAACATA
416..419
ATAC
348..355
ACACACAG




414..418
ACATA
418..419
AC







Basma
18GH203
414..417
ACAT
412..418
CAACATA
348..357
ACACACAGAG(392)






415..418
CATA
353..354
CA





Katerini
18GH208
416..419
ATAC
414..417
ACAT
352..355
ACAG




418..419
AC









Basma
18GH341
414..417
ACAT
412..418
CAACATA
348..357
ACACACAGAG(392)






415..418
CATA
353..354
CA





Katerini
18GH403
414..417
ACAT
414..417
ACAT
348..351
ACAC





Katerini
18GH414
412..418
CAACATA
414..417
ACAT
348..349
AC




415..418
CATA









Katerini
18GH434
413..420
AACATACA
414..417
ACAT
351..357
CACAGAG




417..420
TACA









Katerini
18GH436
412..418
CAACATA
414..421
ACATACAG
346..352
CAACACA




415..416
CA









Katerini
18GH437
414..417
ACAT
414..417
ACAT
348..349
AC






416..417
AT







Katerini
18GH449
414..417
ACAT
413..419
AACATAC
348..349
AC




415..416
CA
418..419
AC







Katerini
18GH706
416..420
ATACA
414..417
ACAT
348..351
ACAC




419..420
CA









Katerini
18GH709
412..418
CAACATA
414..417
ACAT
348..349
AC




417..418
TA
416..417
AT







Katerini
18GH710
414..417
ACAT
414..417
ACAT
351..354
CACA








352..355
ACAG





Katerini
18GH716
416..419
ATAC
417..420
TACA
348..351
ACAC




418..419
AC
417..421
TACAG







Katerini
18GH729
414..417
ACAT
414..417
ACAT
350..357
ACACAGAG








353..354
CA





Katerini
18GH731
414..417
ACAT
414..417
ACAT
348..351
ACAC





Katerini
18GH752
416..419
ATAC
414..417
ACAT
348..349
AC




418..419
AC









Katerini
18GH756
416..419
ATAC
414..417
ACAT
353..354
CA




416..419
ATAC
414..417
ACAT
353..354
CA





Katerini
18GH768
416..419
ATAC
414..417
ACAT
348..351
ACAC





Katerini
18GH771
416..419
ATAC
414..417
ACAT
346..352
CAACACA








349..352
CACA





Katerini
18GH776
409..415
ATTCAAC
411..417
TCAACAT
348..351
ACAC




418..419
AC
414..417
ACAT







Katerini
18GH800
412..418
CAACATA
414..420
ACATACA
348..351
ACAC




415..418
CATA
416..420
ATACA







Katerini
18GH818
414..417
ACAT
409..415
ATTCAAC
348..351
ACAC

















PMT3
PMT4

















Deleted

Deleted



VARIETY
LINE
Position
sequence
Position
sequence






Katerini
18GH125
432..435
ACAC
546..547
AC





434..435
AC








Basma
18GH203
432..435
ACAC
546..549
ACAC





434..435
AC








Katerini
18GH208
432..441
ACACACAGAG(392)
546..553
ACACACAG





435..438
CACA








Basma
18GH341
432..435
ACAC
546..549
ACAC





434..435
AC








Katerini
18GH403
432..435
ACAC








Katerini
18GH414
430..436
CAACACA
546..547
AC






Katerini
18GH434
433..439
CACACAG
546..547
AC





437..438
CA








Katerini
18GH436
432..433
AC
546..547
AC





436..443
ACAGAGAA







445..449
GGTGG







451..463
TTTCCATACACTG








(402)








Katerini
18GH437
433..439
CACACAG
544..553
CAACACACAG(390)





435..438
CACA
551..552
CA






Katerini
18GH449
432..435
ACAC
546..549
ACAC






Katerini
18GH706
432..439
ACACACAG
545..555
AACACACAGAG(391)





435..438
CACA








Katerini
18GH709
432..435
ACAC
546..547
AC






Katerini
18GH710
432..435
ACAC
546..547
AC





434..435
AC








Katerini
18GH716
432..439
ACACACAG
546..549
ACAC





435..438
CACA
548..549
AC






Katerini
18GH729
432..435
ACAC
546..547
AC






Katerini
18GH731
433..433
C
544..553
CAACACACAG(390)





435..439
CACAG








Katerini
18GH752
432..435
ACAC
546..549
ACAC






Katerini
18GH756
433..439
CACACAG
545..555
AACACACAGAG(391)





433..439
CACACAG
545..555
AACACACAGAG(391)





437..438
CA
549..552
CACA






Katerini
18GH768
432..433
AC
544..550
CAACACA







547..550
CACA






Katerini
18GH771
432..435
ACAC
546..547
AC





434..435
AC








Katerini
18GH776
441..441
G
546..549
ACAC







548..549
AC






Katerini
18GH800
432..435
ACAC
541..551
ATTCAACACAC(403)







548..551
ACAC






Katerini
18GH818
432..435
ACAC







434..435
AC
















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 from

Position


LINE
Modification
from ATG

from ATG

from ATG

ATG

from ATG




















CS15
A-deleted
131
A-
132
A-deleted
98
A-deleted
98
A-del
131



T-deleted
262
inserted

T-deleted
196
T-deleted
282
















TABLE 5A







A list of exemplary mutant alleles obtained in the PMT1b 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





Allele

Allele




Deleted
sequence

sequence




sequence
(SEQ ID
Mutant
(SEQ ID



Position
(SEQ ID No.)
No.)
Allele Sequence
No.)
Reference Allele Sequence





131...131
A
23
TGGCATTTCCAAACACCAA
201
TGGCATTTCCAAACACCAAAaCGGGCACCA





ACGGGCACCAGAATGGCACTT

GAATGGCACTT





262...262
T
24
CCAACTCTATTAAGCCTGGT
202
CCAACTCTATTAAGCCTGGTtGGTTTTCAGA





GGTTTTCAGAGTTTAGCGCA

GTTTAGCGCA





409..415
ATTCAAC
25
TTCTGACTTTGGATGGAGCA
203
TTCTGACTTTGGATGGAGCAattcaacATACAG





ATACAGAGAATGGTGGATTT

AGAATGGTGGATTT





411..420
TCAACATACA
26
CTGACTTTGGATGGAGCAA
204
CTGACTTTGGATGGAGCAATtcaacatacaGAGA



(379)

TGAGAATGGTGGATTTCCATA

ATGGTGGATTTCCATA





412..418
CAACATA
27
TGACTTTGGATGGAGCAAT
205
TGACTTTGGATGGAGCAATTcaacataCAGAGA





TCAGAGAATGGTGGATTTCCA

ATGGTGGATTTCCA





412..421
CAACATACAG
28
TGACTTTGGATGGAGCAAT
206
TGACTTTGGATGGAGCAATTcaacatacagAGAA



(380)

TAGAATGGTGGATTTCCATAC

TGGTGGATTTCCATAC





413..419
AACATAC
29
GACTTTGGATGGAGCAATT
207
GACTTTGGATGGAGCAATTCaacatacAGAGAA





CAGAGAATGGTGGATTTCCAT

TGGTGGATTTCCAT





413..420
AACATACA
30
GACTTTGGATGGAGCAATT
208
GACTTTGGATGGAGCAATTCaacatacaGAGAA





CGAGAATGGTGGATTTCCATA

TGGTGGATTTCCATA





413..421
AACATACAG
31
GACTTTGGATGGAGCAATT
209
GACTTTGGATGGAGCAATTCaacatacagAGAA





CAGAATGGTGGATTTCCATAC

TGGTGGATTTCCATAC





413..428
AACATACAGAGA
32
GACTTTGGATGGAGCAATT
210
GACTTTGGATGGAGCAATTCaacatacagagaatgg



ATGG(381)

CTGGATTTCCATACACTGAAA

TGGATTTCCATACACTGAAA





414..417
ACAT
33
ACTTTGGATGGAGCAATTC
211
ACTTTGGATGGAGCAATTCAacatACAGAGA





AtACAGAGAATGGTGGATTT

ATGGTGGATTTCC





CC







414..418
ACATA
34
ACTTTGGATGGAGCAATTC
212
ACTTTGGATGGAGCAATTCAacataCAGAGAA





ACAGAGAATGGTGGATTTCCA

TGGTGGATTTCCA





414..419
ACATAC
35
ACTTTGGATGGAGCAATTC
213
ACTTTGGATGGAGCAATTCAacatacAGAGAA





AAGAGAATGGTGGATTTCCAT

TGGTGGATTTCCAT





414..420
ACATACA
36
ACTTTGGATGGAGCAATTC
214
ACTTTGGATGGAGCAATTCAacatacaGAGAAT





AGAGAATGGTGGATTTCCATA

GGTGGATTTCCATA





414..437
ACATACAGAGAA
37
ACTTTGGATGGAGCAATTC
215
ACTTTGGATGGAGCAATTCAacatacagagaatggt



TGGTGGATTTCC

AATACACTGAAATGATTGT

ggatttccATACACTGAAATGATTGTTC



(382)

TC







415..416
CA
38
CTTTGGATGGAGCAATTCA
216
CTTTGGATGGAGCAATTCAAcaTACAGAGAA





ATACAGAGAATGGTGGATTTC

TGGTGGATTTC





415..418
CATA
39
CTTTGGATGGAGCAATTCA
217
CTTTGGATGGAGCAATTCAAcataCAGAGAAT





ACAGAGAATGGTGGATTTCCA

GGTGGATTTCCA





415..420
CATACA
40
CTTTGGATGGAGCAATTCA
218
CTTTGGATGGAGCAATTCAAcatacaGAGAAT





AGAGAATGGTGGATTTCCATA

GGTGGATTTCCATA





415..421
CATACAG
41
CTTTGGATGGAGCAATTCA
219
CTTTGGATGGAGCAATTCAAcatacagAGAATG





AAGAATGGTGGATTTCCATAC

GTGGATTTCCATAC





416..416
A
42
TTTGGATGGAGCAATTCAA
220
TTTGGATGGAGCAATTCAACaTACAGAGAA





CTACAGAGAATGGTGGATTTC

TGGTGGATTTC





416..418
ATA
43
TTTGGATGGAGCAATTCAA
221
TTTGGATGGAGCAATTCAACataCAGAGAAT





CCAGAGAATGGTGGATTTCCA

GGTGGATTTCCA





416..419
ATAC
44
TTTGGATGGAGCAATTCAA
222
TTTGGATGGAGCAATTCAACatacAGAGAATG





CAGAGAATGGTGGATTTCCAT

GTGGATTTCCAT





416..420
ATACA
45
TTTGGATGGAGCAATTCAA
223
TTTGGATGGAGCAATTCAACatacaGAGAATG





CGAGAATGGTGGATTTCCATA

GTGGATTTCCATA





416..421
ATACAG
46
TTTGGATGGAGCAATTCAA
224
TTTGGATGGAGCAATTCAACatacagAGAATG





CAGAATGGTGGATTTCCATAC

GTGGATTTCCATAC





416..423
ATACAGAG
47
TTTGGATGGAGCAATTCAA
225
TTTGGATGGAGCAATTCAACatacagagAATGG





CAATGGTGGATTTCCATACAC

TGGATTTCCATACAC





417..418
TA
48
TTGGATGGAGCAATTCAAC
226
TTGGATGGAGCAATTCAACAtaCAGAGAATG





ACAGAGAATGGTGGATTTCCA

GTGGATTTCCA





417..420
TACA
49
TTGGATGGAGCAATTCAAC
227
TTGGATGGAGCAATTCAACAtacaGAGAATG





AGAGAATGGTGGATTTCCATA

GTGGATTTCCATA





418..419
AC
50
TGGATGGAGCAATTCAACA
228
TGGATGGAGCAATTCAACATacAGAGAATG





TAGAGAATGGTGGATTTCCAT

GTGGATTTCCAT





418..420
ACA
51
TGGATGGAGCAATTCAACA
229
TGGATGGAGCAATTCAACATacaGAGAATGG





TGAGAATGGTGGATTTCCATA

TGGATTTCCATA





418..421
ACAG
52
TGGATGGAGCAATTCAACA
230
TGGATGGAGCAATTCAACATacagAGAATGG





TAGAATGGTGGATTTCCATAC

TGGATTTCCATAC





418..423
ACAGAG
53
TGGATGGAGCAATTCAACA
231
TGGATGGAGCAATTCAACATacagagAATGGT





TAATGGTGGATTTCCATACAC

GGATTTCCATACAC





419..420
CA
54
GGATGGAGCAATTCAACAT
232
GGATGGAGCAATTCAACATAcaGAGAATGG





AGAGAATGGTGGATTTCCATA

TGGATTTCCATA





427..427
G
55
CAATTCAACATACAGAGAA
233
CAATTCAACATACAGAGAATgGTGGATTTC





TGTGGATTTCCATACACTGA

CATACACTGAA





A
















TABLE 5B







A list of exemplary mutant alleles obtained in the PMT1a gene.


PMT1a














Mutant

Reference





Allele

Allele




Deleted
sequence

sequence




sequence
(SEQ ID
Mutant
(SEQ ID



Position
(SEQ ID No.)
No.)
Allele Sequence
No.)
Reference Allele Sequence





132...132
A inserted
56
GCACTTCCAAACACCAAAACa
234
GCACTTCCAAACACCAAAACGGGCA





GGGCACCAGAATGGCACTTT

CCAGAATGGCACTTT





409..415
ATTCAAC
57
TTCTGACTTTGGATGGAGCAA
235
TTCTGACTTTGGATGGAGCAattcaacAT





TACAGAGAATGGTGGATTT

ACAGAGAATGGTGGATTT





409..420
ATTCAACATACA
58
TTCTGACTTTGGATGGAGCAG
236
TTCTGACTTTGGATGGAGCAattcaacatac



(383)

AGAATGGTGGATTTCCATA

aGAGAATGGTGGATTTCCATA





411..417
TCAACAT
59
CTGACTTTGGATGGAGCAATA
237
CTGACTTTGGATGGAGCAATtcaacatAC





CAGAGAATGGTGGATTTCC

AGAGAATGGTGGATTTCC





411..420
TCAACATACA
60
CTGACTTTGGATGGAGCAATG
238
CTGACTTTGGATGGAGCAATtcaacataca



(384)

AGAATGGTGGATTTCCATA

GAGAATGGTGGATTTCCATA





412..418
CAACATA
61
TGACTTTGGATGGAGCAATTC
239
TGACTTTGGATGGAGCAATTcaacataCA





AGAGAATGGTGGATTTCCA

GAGAATGGTGGATTTCCA





412..421
CAACATACAG
62
TGACTTTGGATGGAGCAATTA
240
TGACTTTGGATGGAGCAATTcaacatacag



(385)

GAATGGTGGATTTCCATAC

AGAATGGTGGATTTCCATAC





413..419
AACATAC
63
GACTTTGGATGGAGCAATTCA
241
GACTTTGGATGGAGCAATTCaacatacA





GAGAATGGTGGATTTCCAT

GAGAATGGTGGATTTCCAT





413..420
AACATACA
64
GACTTTGGATGGAGCAATTCG
242
GACTTTGGATGGAGCAATTCaacatacaG





AGAATGGTGGATTTCCATA

AGAATGGTGGATTTCCATA





413..421
AACATACAG
65
GACTTTGGATGGAGCAATTCA
243
GACTTTGGATGGAGCAATTCaacatacag





GAATGGTGGATTTCCATAC

AGAATGGTGGATTTCCATAC





413..422
AACATACAGA
66
GACTTTGGATGGAGCAATTCG
244
GACTTTGGATGGAGCAATTCaacatacag



(386)

AATGGTGGATTTCCATACA

aGAATGGTGGATTTCCATACA





413..428
AACATACAGAG
67
GACTTTGGATGGAGCAATTCT
245
GACTTTGGATGGAGCAATTCaacatacag



AATGG(387)

GGATTTCCATACACTGAAA

agaatggTGGATTTCCATACACTGAAA





414..415
AC
68
ACTTTGGATGGAGCAATTCAA
246
ACTTTGGATGGAGCAATTCAacATAC





TACAGAGAATGGTGGATTT

AGAGAATGGTGGATTT





414..417
ACAT
69
ACTTTGGATGGAGCAATTCAA
247
ACTTTGGATGGAGCAATTCAacatACA





CAGAGAATGGTGGATTTCC

GAGAATGGTGGATTTCC





414..419
ACATAC
70
ACTTTGGATGGAGCAATTCAA
248
ACTTTGGATGGAGCAATTCAacatacAG





GAGAATGGTGGATTTCCAT

AGAATGGTGGATTTCCAT





414..420
ACATACA
71
ACTTTGGATGGAGCAATTCAG
249
ACTTTGGATGGAGCAATTCAacatacaG





AGAATGGTGGATTTCCATA

AGAATGGTGGATTTCCATA





414..421
ACATACAG
72
ACTTTGGATGGAGCAATTCAA
250
ACTTTGGATGGAGCAATTCAacatacagA





GAATGGTGGATTTCCATAC

GAATGGTGGATTTCCATAC





414..423
ACATACAGAG
73
ACTTTGGATGGAGCAATTCAA
251
ACTTTGGATGGAGCAATTCAacatacaga



(388)

ATGGTGGATTTCCATACAC

gAATGGTGGATTTCCATACAC





415..415
C
74
CTTTGGATGGAGCAATTCAAA
252
CTTTGGATGGAGCAATTCAAcATACA





TACAGAGAATGGTGGATTT

GAGAATGGTGGATTT





415..416
CA
75
CTTTGGATGGAGCAATTCAAT
253
CTTTGGATGGAGCAATTCAAcaTACA





ACAGAGAATGGTGGATTTC

GAGAATGGTGGATTTC





415..418
CATA
76
CTTTGGATGGAGCAATTCAAC
254
CTTTGGATGGAGCAATTCAAcataCAG





AGAGAATGGTGGATTTCCA

AGAATGGTGGATTTCCA





415..419
CATAC
77
CTTTGGATGGAGCAATTCAAA
255
CTTTGGATGGAGCAATTCAAcatacAGA





GAGAATGGTGGATTTCCAT

GAATGGTGGATTTCCAT





415..421
CATACAG
78
CTTTGGATGGAGCAATTCAAA
256
CTTTGGATGGAGCAATTCAAcatacagA





GAATGGTGGATTTCCATAC

GAATGGTGGATTTCCATAC





416..417
AT
79
TTTGGATGGAGCAATTCAACtA
257
TTTGGATGGAGCAATTCAACatACAGA





CAGAGAATGGTGGATTTCC

GAATGGTGGATTTCC





416..419
ATAC
80
TTTGGATGGAGCAATTCAACA
258
TTTGGATGGAGCAATTCAACatacAGA





GAGAATGGTGGATTTCCAT

GAATGGTGGATTTCCAT





416..420
ATACA
81
TTTGGATGGAGCAATTCAACG
259
TTTGGATGGAGCAATTCAACatacaGAG





AGAATGGTGGATTTCCATA

AATGGTGGATTTCCATA





416..421
ATACAG
82
TTTGGATGGAGCAATTCAACA
260
TTTGGATGGAGCAATTCAACatacagAG





GAATGGTGGATTTCCATAC

AATGGTGGATTTCCATAC





416..422
ATACAGA
83
TTTGGATGGAGCAATTCAACG
261
TTTGGATGGAGCAATTCAACatacagaG





AATGGTGGATTTCCATACA

AATGGTGGATTTCCATACA





416..429
ATACAGAGAAT
84
TTTGGATGGAGCAATTCAACG
262
TTTGGATGGAGCAATTCAACatacagaga



GGT(389)

GATTTCCATACACTGAAAT

atggtGGATTTCCATACACTGAAAT





417..417
T
85
TTGGATGGAGCAATTCAACAA
263
TTGGATGGAGCAATTCAACAtACAGA





CAGAGAATGGTGGATTTCC

GAATGGTGGATTTCC





417..418
TA
86
TTGGATGGAGCAATTCAACAC
264
TTGGATGGAGCAATTCAACAtaCAGA





AGAGAATGGTGGATTTCCA

GAATGGTGGATTTCCA





417..419
TAC
87
TTGGATGGAGCAATTCAACAA
265
TTGGATGGAGCAATTCAACAtacAGAG





GAGAATGGTGGATTTCCAT

AATGGTGGATTTCCAT





417..420
TACA
88
TTGGATGGAGCAATTCAACAG
266
TTGGATGGAGCAATTCAACAtacaGAG





AGAATGGTGGATTTCCATA

AATGGTGGATTTCCATA





417..421
TACAG
89
TTGGATGGAGCAATTCAACAA
267
TTGGATGGAGCAATTCAACAtacagAG





GAATGGTGGATTTCCATAC

AATGGTGGATTTCCATAC





417..422
TACAGA
90
TTGGATGGAGCAATTCAACAG
268
TTGGATGGAGCAATTCAACAtacagaGA





AATGGTGGATTTCCATACA

ATGGTGGATTTCCATACA





417.423
TACAGAG
91
TTGGATGGAGCAATTCAACAA
269
TTGGATGGAGCAATTCAACAtacagagA





ATGGTGGATTTCCATACAC

ATGGTGGATTTCCATACAC





418..419
AC
92
TGGATGGAGCAATTCAACATA
270
TGGATGGAGCAATTCAACATacAGAG





GAGAATGGTGGATTTCCAT

AATGGTGGATTTCCAT





418..421
ACAG
93
TGGATGGAGCAATTCAACATA
271
TGGATGGAGCAATTCAACATacagAGA





GAATGGTGGATTTCCATAC

ATGGTGGATTTCCATAC





418..424
ACAGAGA
94
TGGATGGAGCAATTCAACATA
272
TGGATGGAGCAATTCAACATacagagaA





TGGTGGATTTCCATACACT

TGGTGGATTTCCATACACT





419..420
CA
95
GGATGGAGCAATTCAACATAG
273
GGATGGAGCAATTCAACATAcaGAGA





AGAATGGTGGATTTCCATA

ATGGTGGATTTCCATA





419..421
CAG
96
GGATGGAGCAATTCAACATAA
274
GGATGGAGCAATTCAACATAcagAGA





GAATGGTGGATTTCCATAC

ATGGTGGATTTCCATAC





424..425
AA
97
GAGCAATTCAACATACAGAGT
275
GAGCAATTCAACATACAGAGaaTGGT





GGTGGATTTCCATACACTG

GGATTTCCATACACTG
















TABLE 5C







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


PMT2














Mutant

Reference





Allele

Allele




Deleted
sequence

sequence




sequence
(SEQ ID
Mutant
(SEQ ID



Position
(SEQ ID No.)
No.)
Allele Sequence
No.)
Reference Allele Sequence





98...98
A
 98
TGGCACTTCCAAACACCA
276
TGGCACTTCCAAACACCAAAaCG





AACGGCCACAAGAATGGG

GCCACAAGAATGGGACTT





ACTT







196...619
T
 99
CCAATTGTATTAAGCCTGG
277
CCAATTGTATTAAGCCTGGTtGG





TGGTTTTCAGAGTTTAGCGCA

TTTTCAGAGTTTAGCGCA





346..350
CAACA
100
TGACTTTGGATGGAGCAAT
278
TGACTTTGGATGGAGCAATTcaac





TCACAGAGAATGGTGGATTTC

aCACAGAGAATGGTGGATTTC





346..352
CAACACA
101
TGACTTTGGATGGAGCAAT
279
TGACTTTGGATGGAGCAATTcaac





TCAGAGAATGGTGGATTTCCA

acaCAGAGAATGGTGGATTTCCA





346..355
CAACACACAG
102
TGACTTTGGATGGAGCAAT
280
TGACTTTGGATGGAGCAATTcaac



(390)

TAGAATGGTGGATTTCCATAC

acacagAGAATGGTGGATTTCCATAC





347..354
AACACACA
103
GACTTTGGATGGAGCAATT
281
GACTTTGGATGGAGCAATTCaaca





CGAGAATGGTGGATTTCCATA

cacaGAGAATGGTGGATTTCCATA





347..357
AACACACAGAG
104
GACTTTGGATGGAGCAATT
282
GACTTTGGATGGAGCAATTCaaca



(391)

CAATGGTGGATTTCCATACAC

cacagagAATGGTGGATTTCCATACAC





348..349
AC
105
ACTTTGGATGGAGCAATTC
283
ACTTTGGATGGAGCAATTCAacA





AACACAGAGAATGGTGGATTT

CACAGAGAATGGTGGATTT





348..351
ACAC
106
ACTTTGGATGGAGCAATTC
284
ACTTTGGATGGAGCAATTCAacac





AACAGAGAATGGTGGATTTCC

ACAGAGAATGGTGGATTTCC





348..352
ACACA
107
ACTTTGGATGGAGCAATTC
285
ACTTTGGATGGAGCAATTCAacac





ACAGAGAATGGTGGATTTCCA

aCAGAGAATGGTGGATTTCCA





348..353
ACACAC
108
ACTTTGGATGGAGCAATTC
286
ACTTTGGATGGAGCAATTCAacac





AAGAGAATGGTGGATTTCCAT

acAGAGAATGGTGGATTTCCAT





348..354
ACACACA
109
ACTTTGGATGGAGCAATTC
287
ACTTTGGATGGAGCAATTCAacac





AGAGAATGGTGGATTTCCATA

acaGAGAATGGTGGATTTCCATA





348..355
ACACACAG
110
ACTTTGGATGGAGCAATTC
288
ACTTTGGATGGAGCAATTCAacac





AAGAATGGTGGATTTCCATAC

acagAGAATGGTGGATTTCCATAC





348..357
ACACACAGAG
111
ACTTTGGATGGAGCAATTC
289
ACTTTGGATGGAGCAATTCAacac



(392)

AAATGGTGGATTTCCATACAC

acagagAATGGTGGATTTCCATACAC





349..349
C
112
CTTTGGATGGAGCAATTCA
290
CTTTGGATGGAGCAATTCAAcAC





AACACAGAGAATGGTGGATTT

ACAGAGAATGGTGGATTT





349..350
CA
113
CTTTGGATGGAGCAATTCA
291
CTTTGGATGGAGCAATTCAAcaC





ACACAGAGAATGGTGGATTTC

ACAGAGAATGGTGGATTTC





349..352
CACA
114
CTTTGGATGGAGCAATTCA
292
CTTTGGATGGAGCAATTCAAcaca





ACAGAGAATGGTGGATTT

CAGAGAATGGTGGATTTCCA





CCA







349..355
CACACAG
115
CTTTGGATGGAGCAATTCA
293
CTTTGGATGGAGCAATTCAAcaca





AAGAATGGTGGATTTCCATAC

cagAGAATGGTGGATTTCCATAC





350..351
AC
116
TTTGGATGGAGCAATTCAA
294
TTTGGATGGAGCAATTCAACacA





CACAGAGAATGGTGGATTTCC

CAGAGAATGGTGGATTTCC





350..353
ACAC
117
TTTGGATGGAGCAATTCAA
295
TTTGGATGGAGCAATTCAACacac





CAGAGAATGGTGGATTTCCAT

AGAGAATGGTGGATTTCCAT





350..354
ACACA
118
TTTGGATGGAGCAATTCAA
296
TTTGGATGGAGCAATTCAACacac





CGAGAATGGTGGATTTCCATA

aGAGAATGGTGGATTTCCATA





350..355
ACACAG
119
TTTGGATGGAGCAATTCAA
297
TTTGGATGGAGCAATTCAACacac





CAGAATGGTGGATTTCCATAC

agAGAATGGTGGATTTCCATAC





350..357
ACACAGAG
120
TTTGGATGGAGCAATTCAA
298
TTTGGATGGAGCAATTCAACacac





CAATGGTGGATTTCCATACAC

agagAATGGTGGATTTCCATACAC





350..363
ACACAGAGA
121
TTTGGATGGAGCAATTCAA
299
TTTGGATGGAGCAATTCAACacac



ATGGT(393)

CGGATTTCCATACACTGAAAT

agagaatggtGGATTTCCATACACTG







AAAT





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

gagaatggTGGATTTCCATACACTGA





AAA

AA





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.


PT3














Mutant

Reference





Allele

Allele




Deleted
sequence

sequence




sequence
(SEQ ID
Mutant
(SEQ ID



Position
(SEQ ID No.)
No.)
Allele Sequence
No.)
Reference Allele Sequence





98...98
A
133
TGGCACTTCCAAACACCAAACGGC
311
TGGCACTTCCAAACACCAAAaCGGCCA





CACCAGAATGGCACTT

CCAGAATGGCACTT





280...280
T
134
CCAACTCTATTAAGCCTGGTGGTTT
312
CCAACTCTATTAAGCCTGGTtGGTTTTC





TCAGAGTTTAGCGCA

AGAGTTTAGCGCA





413..414
CT
135
AACATATGGGAAGGTTCTGATTGG
313
AACATATGGGAAGGTTCTGActTTGGAT





ATGGAGCAATTCAACA

GGAGCAATTCAACA





418..419
GA
136
ATGGGAAGGTTCTGACTTTGTGGA
314
ATGGGAAGGTTCTGACTTTGgaTGGAGC





GCAATTCAACACACAG

AATTCAACACACAG





426..427
AA
137
GTTCTGACTTTGGATGGAGCTTCA
315
GTTCTGACTTTGGATGGAGCaaTTCAAC





ACACACAGAGAATGGT

ACACAGAGAATGGT





429..435
TCAACAC
138
CTGACTTTGGATGGAGCAATACAG
316
CTGACTTTGGATGGAGCAATtcaacacACA





AGAATGGTGGATTTCC

GAGAATGGTGGATTTCC





429..438
TCAACACACA
139
CTGACTTTGGATGGAGCAATGAGA
317
CTGACTTTGGATGGAGCAATtcaacacacaG



(395)

ATGGTGGATTTCCATA

AGAATGGTGGATTTCCATA





429..439
TCAACACACAG
140
CTGACTTTGGATGGAGCAATAGAA
318
CTGACTTTGGATGGAGCAATtcaacacacag



(396)

TGGTGGATTTCCATAC

AGAATGGTGGATTTCCATAC





430..436
CAACACA
141
TGACTTTGGATGGAGCAATTCAGA
319
TGACTTTGGATGGAGCAATTcaacacaCAG





GAATGGTGGATTTCCA

AGAATGGTGGATTTCCA





431..431
A
142
GACTTTGGATGGAGCAATTCACAC
320
GACTTTGGATGGAGCAATTCaACACACA





ACAGAGAATGGTGGAT

GAGAATGGTGGAT





431..432
AA
143
GACTTTGGATGGAGCAATTCCACA
321
GACTTTGGATGGAGCAATTCaaCACACA





CAGAGAATGGTGGATT

GAGAATGGTGGATT





431..438
AACACACA
144
GACTTTGGATGGAGCAATTCGAGA
322
GACTTTGGATGGAGCAATTCaacacacaGA





ATGGTGGATTTCCATA

GAATGGTGGATTTCCATA





431..441
AACACACAGAG
145
GACTTTGGATGGAGCAATTCAATG
323
GACTTTGGATGGAGCAATTCaacacacagag



(397)

GTGGATTTCCATACAC

AATGGTGGATTTCCATACAC





432..433
AC
146
ACTTTGGATGGAGCAATTCAACAC
324
ACTTTGGATGGAGCAATTCAacACACAG





AGAGAATGGTGGATTT

AGAATGGTGGATTT





432..435
ACAC
147
ACTTTGGATGGAGCAATTCAACAG
325
ACTTTGGATGGAGCAATTCAacacACAGA





AGAATGGTGGATTTCC

GAATGGTGGATTTCC





432..437
ACACAC
148
ACTTTGGATGGAGCAATTCAAGAG
326
ACTTTGGATGGAGCAATTCAacacacAGA





AATGGTGGATTTCCAT

GAATGGTGGATTTCCAT





432..439
ACACACAG
149
ACTTTGGATGGAGCAATTCAAGAA
327
ACTTTGGATGGAGCAATTCAacacacagAG





TGGTGGATTTCCATAC

AATGGTGGATTTCCATAC





432...441
ACACACAGAG
150
ACTTTGGATGGAGCAATTCAAATG
328
ACTTTGGATGGAGCAATTCAacacacagagA



(398)

GTGGATTTCCATACAC

ATGGTGGATTTCCATACAC





432..446
ACACACAGAG
151
ACTTTGGATGGAGCAATTCATGGA
329
ACTTTGGATGGAGCAATTCAacacacagaga



AATGG(399)

TTTCCATACACTGAAA

atggTGGATTTCCATACACTGAAA





432..448
ACACACAGAG
152
ACTTTGGATGGAGCAATTCAGATT
330
ACTTTGGATGGAGCAATTCAacacacagaga



AATGGTG(400)

TCCATACACTGAAATG

atggtgGATTTCCATACACTGAAATG





433..433
C
153
CTTTGGATGGAGCAATTCAAACAC
331
CTTTGGATGGAGCAATTCAAcACACAG





AGAGAATGGTGGATTT

AGAATGGTGGATTT





433..436
CACA
154
CTTTGGATGGAGCAATTCAACAGA
332
CTTTGGATGGAGCAATTCAAcacaCAGAG





GAATGGTGGATTTCCA

AATGGTGGATTTCCA





433..437
CACAC
155
CTTTGGATGGAGCAATTCAAAGAG
333
CTTTGGATGGAGCAATTCAAcacacAGAG





AATGGTGGATTTCCAT

AATGGTGGATTTCCAT





433..439
CACACAG
156
CTTTGGATGGAGCAATTCAAAGAA
334
CTTTGGATGGAGCAATTCAAcacacagAGA





TGGTGGATTTCCATAC

ATGGTGGATTTCCATAC





434..435
AC
157
TTTGGATGGAGCAATTCAACACAG
335
TTTGGATGGAGCAATTCAACacACAGAG





AGAATGGTGGATTTCC

AATGGTGGATTTCC





434..437
ACAC
158
TTTGGATGGAGCAATTCAACAGAG
336
TTTGGATGGAGCAATTCAACacacAGAG





AATGGTGGATTTCCAT

AATGGTGGATTTCCAT





434..438
ACACA
159
TTTGGATGGAGCAATTCAACGAGA
337
TTTGGATGGAGCAATTCAACacacaGAGA





ATGGTGGATTTCCATA

ATGGTGGATTTCCATA





435..436
CA
160
TTGGATGGAGCAATTCAACACAGA
338
TTGGATGGAGCAATTCAACAcaCAGAGA





GAATGGTGGATTTCCA

ATGGTGGATTTCCA





435..438
CACA
161
TTGGATGGAGCAATTCAACAGAGA
339
TTGGATGGAGCAATTCAACAcacaGAGA





ATGGTGGATTTCCATA

ATGGTGGATTTCCATA





435..439
CACAG
162
TTGGATGGAGCAATTCAACAAGAA
340
TTGGATGGAGCAATTCAACAcacagAGAA





TGGTGGATTTCCATAC

TGGTGGATTTCCATAC





435..447
CACAGAGAAT
163
TTGGATGGAGCAATTCAACAGGAT
341
TTGGATGGAGCAATTCAACAcacagagaatg



GGT(401)

TTCCATACACTGAAAT

gtGGATTTCCATACACTGAAAT





436..437
AC
164
TGGATGGAGCAATTCAACACAGAG
342
TGGATGGAGCAATTCAACACacAGAGA





AATGGTGGATTTCCAT

ATGGTGGATTTCCAT





436..439
ACAG
165
TGGATGGAGCAATTCAACACAGAA
343
TGGATGGAGCAATTCAACACacagAGAA





TGGTGGATTTCCATAC

TGGTGGATTTCCATAC





436..443
ACAGAGAA
166
TGGATGGAGCAATTCAACACTGGT
344
TGGATGGAGCAATTCAACACacagagaaTG





GGATTTCCATACACTG

GTGGATTTCCATACACTG





437..438
CA
167
GGATGGAGCAATTCAACACAGAG
345
GGATGGAGCAATTCAACACAcaGAGAA





AATGGTGGATTTCCATA

TGGTGGATTTCCATA





440..442
AGA
168
TGGAGCAATTCAACACACAGATGG
346
TGGAGCAATTCAACACACAGagaATGGT





TGGATTTCCATACACT

GGATTTCCATACACT





440..443
AGAA
169
TGGAGCAATTCAACACACAGTGGT
347
TGGAGCAATTCAACACACAGagaaTGGT





GGATTTCCATACACTG

GGATTTCCATACACTG





441..441
G
170
GGAGCAATTCAACACACAGAAATG
348
GGAGCAATTCAACACACAGAgAATGGT





GTGGATTTCCATACAC

GGATTTCCATACAC





445..449
GGTGG
171
CAATTCAACACACAGAGAATATTT
349
CAATTCAACACACAGAGAATggtggATTT





CCATACACTGAAATGA

CCATACACTGAAATGA





451..463
TTTCCATACAC
172
AACACACAGAGAATGGTGGAAAA
350
AACACACAGAGAATGGTGGAtttccatacact



TG(402)

TGATTGTTCATCTTCCA

gAAATGATTGTTCATCTTCCA
















TABLE 5E







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


PMT4














Mutant

Reference





Allele

Allele




Deleted
sequence

sequence




sequence
(SEQ ID
Mutant
(SEQ ID



Position
(SEQ ID No.)
No.)
Allele Sequence
No.)
Reference Allele Sequence





131...131
A
173
CGGCACTTCCAAACACCAAACGGCCA
351
CGGCACTTCCAAACACCAAAaCGGCCAC





CCATAATGGCACTT

CATAATGGCACTT





541..551
ATTCAACACAC
174
TTTTGACTTTGGATGGAGCAAGAGAAT
352
TTTTGACTTTGGATGGAGCAattcaacacacA



(403)

GGTGGATTTCCAT

GAGAATGGTGGATTTCCAT





543..554
TCAACACACAGA
175
TTGACTTTGGATGGAGCAATGAATGGT
353
TTGACTTTGGATGGAGCAATtcaacacacaga



(404)

GGATTTCCATACA

GAATGGTGGATTTCCATACA





544..550
CAACACA
176
TGACTTTGGATGGAGCAATTCAGAGA
354
TGACTTTGGATGGAGCAATTcaacacaCAG





ATGGTGGATTTCCA

AGAATGGTGGATTTCCA





544..553
CAACACACAG
177
TGACTTTGGATGGAGCAATTAGAATGG
355
TGACTTTGGATGGAGCAATTcaacacacagA



(405)

TGGATTTCCATAC

GAATGGTGGATTTCCATAC





545..555
AACACACAGAG
178
GACTTTGGATGGAGCAATTCAATGGTG
356
GACTTTGGATGGAGCAATTCaacacacagag



(406)

GATTTCCATACAC

AATGGTGGATTTCCATACAC





545..557
AACACACAGAGAA
179
GACTTTGGATGGAGCAATTCTGGTGGA
357
GACTTTGGATGGAGCAATTCaacacacagaga



(407)

TTTCCATACACTG

aTGGTGGATTTCCATACACTG





546..547
AC
180
ACTTTGGATGGAGCAATTCAACACAG
358
ACTTTGGATGGAGCAATTCAacACACAG





AGAATGGTGGATTT

AGAATGGTGGATTT





546..549
ACAC
181
ACTTTGGATGGAGCAATTCAACAGAG
359
ACTTTGGATGGAGCAATTCAacacACAGA





AATGGTGGATTTCC

GAATGGTGGATTTCC





546..551
ACACAC
182
ACTTTGGATGGAGCAATTCAAGAGAA
360
ACTTTGGATGGAGCAATTCAacacacAGAG





TGGTGGATTTCCAT

AATGGTGGATTTCCAT





546..552
ACACACA
183
ACTTTGGATGGAGCAATTCAGAGAAT
361
ACTTTGGATGGAGCAATTCAacacacaGAG





GGTGGATTTCCATA

AATGGTGGATTTCCATA





546..553
ACACACAG
184
ACTTTGGATGGAGCAATTCAAGAATG
362
ACTTTGGATGGAGCAATTCAacacacagAG





GTGGATTTCCATAC

AATGGTGGATTTCCATAC





547..550
CACA
185
CTTTGGATGGAGCAATTCAACAGAGA
363
CTTTGGATGGAGCAATTCAAcacaCAGAG





ATGGTGGATTTCCA

AATGGTGGATTTCCA





547..551
CACAC
186
CTTTGGATGGAGCAATTCAAAGAGAA
364
CTTTGGATGGAGCAATTCAAcacacAGAG





TGGTGGATTTCCAT

AATGGTGGATTTCCAT





548..549
AC
187
TTTGGATGGAGCAATTCAACACAGAG
365
TTTGGATGGAGCAATTCAACacACAGAG





AATGGTGGATTTCC

AATGGTGGATTTCC





548..551
ACAC
188
TTTGGATGGAGCAATTCAACAGAGAA
366
TTTGGATGGAGCAATTCAACacacAGAGA





TGGTGGATTTCCAT

ATGGTGGATTTCCAT





548..552
ACACA
189
TTTGGATGGAGCAATTCAACGAGAAT
367
TTTGGATGGAGCAATTCAACacacaGAGA





GGTGGATTTCCATA

ATGGTGGATTTCCATA





549..552
CACA
190
TTGGATGGAGCAATTCAACAGAGAAT
368
TTGGATGGAGCAATTCAACAcacaGAGAA





GGTGGATTTCCATA

TGGTGGATTTCCATA





549..553
CACAG
191
TTGGATGGAGCAATTCAACAAGAATG
369
TTGGATGGAGCAATTCAACAcacagAGAA





GTGGATTTCCATAC

TGGTGGATTTCCATAC





550..551
AC
192
TGGATGGAGCAATTCAACACAGAGAA
370
TGGATGGAGCAATTCAACACacAGAGAA





TGGTGGATTTCCAT

TGGTGGATTTCCAT





550..552
ACA
193
TGGATGGAGCAATTCAACACGAGAAT
371
TGGATGGAGCAATTCAACACacaGAGAA





GGTGGATTTCCATA

TGGTGGATTTCCATA





550..553
ACAG
194
TGGATGGAGCAATTCAACACAGAATG
372
TGGATGGAGCAATTCAACACacagAGAAT





GTGGATTTCCATAC

GGTGGATTTCCATAC





550..556
ACAGAGA
195
TGGATGGAGCAATTCAACACATGGTG
373
TGGATGGAGCAATTCAACACacagagaATG





GATTTCCATACACT

GTGGATTTCCATACACT





551..552
CA
196
GGATGGAGCAATTCAACACAGAGAAT
374
GGATGGAGCAATTCAACACAcaGAGAAT





GGTGGATTTCCATA

GGTGGATTTCCATA





554..554
A
197
TGGAGCAATTCAACACACAGGAATGG
375
TGGAGCAATTCAACACACAGaGAATGGT





TGGATTTCCATACA

GGATTTCCATACA





558..563
TGGTGG
198
GCAATTCAACACACAGAGAAATTTCC
376
GCAATTCAACACACAGAGAAtggtggATTT





ATACACTGAAATGA

CCATACACTGAAATGA





565..566
TT
199
AACACACAGAGAATGGTGGATCCATA
377
AACACACAGAGAATGGTGGAttTCCATAC





CACTGAAATGATTG

ACTGAAATGATTG





569..572
CATA
200
CACAGAGAATGGTGGATTTCCACTGA
378
CACAGAGAATGGTGGATTTCcataCACTG





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 compromising 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 can be performed to obtain specific PMT mutation combinations. Tables 8A to 8E 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 5A to 5E.


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
















Total



Variety
Plant ID
Nicotine
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
















Total



Variety
Plant ID
Nicotine
Alkaloids















NLM Control
18GH126
2.0844
2.1734




18GH7
3.3504
3.5136



NLM Edited
18GH10
0.001
1.14




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.











Total



Nicotine
Alkaloids












K326 Control
1.880
1.982


K326 quintuple pmt mutant
0.008
0.429


Relative change
0.44%
21.65%


TN90 Control
2.395
2.538


TN90 quintuple pmt mutant
0.005
0.655


Relative change
0.22%
25.80%


NLM Control
2.717
2.844


NLM quintuple pmt mutant
0.006
0.927


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 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





GATGGAGCAATTCAACA
Y
Y





TACAGA







(SEQ ID NO: 730)










GATGGAGCAATTCAACA


Y
Y
Y


CACAGA







(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 PMT1b;


SEQ ID NO: 7 for PMT1a; SEQ ID NO: 8 for PMT2; SEQ ID NO: 9 for PMT3; SEQ ID NO: 10 for


PMT4). SEQ ID Numbers are assigned and shown for sequences of more than 10 nucleotides.





















PMT1a
PMT1b
PMT2














Geno-

Deleted

Deleted

Deleted



type
Line
Sequence
Position
Sequence
Position
Sequence
Position





BASMA
CS107
CAACATA
412.418
ACAT
414..417
AC
348..349





BASMA
CS106
ACAT
414..417
ACAT
414..417
AC
348..349





K326
CS115
TCAACATACA
411..420
ACAT
414..417
AC
348..349




(SEQ ID NO:









379)










K326
18GH2162
ACAT
414..417
ACAT
414..417
ACACACAG
348..355





K326
CS111
ACAT
414..417
ACAT
414..417
AC
348..349





K326
CS112
CATACAG
415..421
AC
418..419
AC
348..349





K326
17GH1678-
CATACAG
415..421
AC
418..419
AC
348..349



60











K326
CS131
ACAT
414..417
ACAT
414..417
ACAC
348..351





KATERINI
CS164
AT
416..417
CAACATA
412..418
AC
348..349





KATERINI
CS163
ACAT
414..417
AT
416..417
AC
348..349





KATERINI
CS146
GAGCAATTCAA
404..422
ACAT
414..417
AC
348..349




CATACAGA









(SEQ ID









NO: 408)










KATERINI
CS147
ACAT
414..417
CAACATA
412..418
AC
348..349





KATERINI
CS150
AT
416..417
ACAT
414..417
AC
348..349





KATERINI
CS151
AT
416..417
ACAT
414..417
AC
348..349





KATERINI
CS148
ACAT
414..417
ACAT
414..417
AC
348..349


KATERINI
CS149
ACAT
414..417
ACAT
414..417
AC
348..349


KATERINI
CS152
AC
418..419
ACAT
414..417
AC
348..349


KATERINI
CS153
CAACAT
412.418
AC
414..415
AC
348..349




A







KATERINI
CS102
ACAT
414..417
AACAT
413..417
ACACAC
348..355








AG



KATERINI
CS103
AC
418..419
CAACAT
412. .418
ACACAC
348..355






A

AG



TN90
CS143
TACAGA
417..423
ACAT
414..417
AC
348..349




G







TN90
18GH2169
AC
418..419
ACAT
414..417
ACAC
348..351


TN90
CS120
ACAT
414..417
ACAG
418..421
ACAC
348..351


TN90
17GH1698-
ACAT
414..417
ACAT
414..417
AC
348..349



22








TN90
17GH1700-
ACAT
414..417
AACAT
413..417
AC
348..349



13








TN90
17GH1702-
ACAT
414..417
ACAT
414..417
AC
348..349



17








TN90
18GH2171
ACAT
414..417
ACAT
414..417
ACAC
348..351


TN90
CS165
ACAT
414..417
ACAT
414..417
ACAC
348..351


TN90
CS118
ACAT
414..417
ACAT
414..417
AC
348..349


TN90
CS133
GGAGCA
403..415
CAACAT
412..421
ACAC
348..351




ATTCAAC

ACAG







(SEQ ID

(SEQ ID







NO: 409)

NO: 380)





TN90
17GH1737-
CA
415..416
ACAT
414..417
ACAC
348..351



24








IZMIR
18GH2254-
CAACAT
412..418
ATAGAG
416..417
ACAC
348..351



7
A

AA
&









420..425



















PMT3
PMT4


















Deleted

Deleted







Sequence
Position
Sequence
Position









ACAC
432..435
ACAC
546..549






ACAC
432..435
ACAC
546..549






CACAC
433..437
ACACA
548..552






AC
432..433
ACACA
548..552






ACAC
432..435
CACAC
547..551






ACAC
432..435
CACAC
547..551






ACAC
432..435
ACAC
546..549






AACACACAG
431..439
ACAC
546..549






ACAC
432..435
AC
546..547






ACAC
432..435
AC
546..547






CAACACA
430..436
AC
546..547






CAACACA
430..436
AC
546..547






CACACAG
433..439
AC
546..547






ACAC
432..435
CAACAC
544..553








ACAG









(SEQ ID









NO: 390)







CACACAG
433..439
AC
546..547






ACAC
432..435
CAACAC
544..553








ACAG









(SEQ ID









NO: 390)







ACAC
432..435
ACAC
546..549






ACAC
432..435
ACAC
546..549






AC
432..433
AC
546..547






AC
432..433
AC
546..547






AC
432..433
ACAC
546..549






ACAC
432..435
AC
546..547






AC
432..433
AC
546..547






ACAG
436..439
AC
546..547






AC
432..433
ACAC
546..549






AC
432..433
CACAG
549..553






CAACACA
430..436
AC
546..547






ACAC
432..435
ACAC
546..549






AC
432..433
ACAC
546..549






AC
432..433
AC
546..547






AC
432..433
AC
546..547






ACAC
432..435
ACACAC
546..553








AG
















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 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: 6) denote which nucleotides are 


deleted in the mutant allele.















Mutant

Reference





Allele

Allele SEQ


Genotype
Line
Mutant Allele Sequence
SEQ ID NO.
Reference Allele Sequence
ID NO.





BASMA
CS107
TCAGCAACTTATGGGAAGGTTCTGACT
410
TCAGCAACTTATGGGAAGGTTCTGACT
442




TTGGATGGAGCAATTCAGAGAATGGTG

TTGGATGGAGCAATTCAacatacaGAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTA

ATTGTTCATCTA






BASMA
CS106
TCAGCAACTTATGGGAAGGTTCTGACT
411
TCAGCAACTTATGGGAAGGTTCTGACT
443




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






K326
CS115
TCAGCAACTTATGGGAAGGTTCTGACT
412
TCAGCAACTTATGGGAAGGTTCTGACT
444




TTGGATGGAGCAATGAGAATGGTGGAT

TTGGATGGAGCAATtcaacatacaGAG





TTCCATACACTGAAATGATTGTTCATC

AATGGTGGATTTCCATACACTGAAATG





TA

ATTGTTCATCTA






K326
18GH2162
TCAGCAACTTATGGGAAGGTTCTGACT
413
TCAGCAACTTATGGGAAGGTTCTGACT
445




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






K326
CS111
TCAGCAACTTATGGGAAGGTTCTGACT
414
TCAGCAACTTATGGGAAGGTTCTGACT
446




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






K326
CS112
TCAGCAACTTATGGGAAGGTTCTGACT
415
TCAGCAACTTATGGGAAGGTTCTGACT
447




TTGGATGGAGCAATTCAAAGAATGGTG

TTGGATGGAGCAATTCAAcatacagAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTA

ATTGTTCATCTATCAAAGAATGG






K326
17GH1678-60
TCAGCAACTTATGGGAAGGTTCTGACT
416
TCAGCAACTTATGGGAAGGTTCTGACT
448




TTGGATGGAGCAATTCAAAGAATGGTG

TTGGATGGAGCAATTCAAcatacagAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTA

ATTGTTCATCTATCAAAGAATGG






K326
CS131
TCAGCAACTTATGGGAAGGTTCTGACT
417
TCAGCAACTTATGGGAAGGTTCTGACT
449




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






KATERINI
CS164
TCAGCAACTTATGGGAAGGTTCTGACT
418
TCAGCAACTTATGGGAAGGTTCTGACT
450




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAACatACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






KATERINI
CS163
TCAGCAACTTATGGGAAGGTTCTGACT
419
TCAGCAACTTATGGGAAGGTTCTGACT
451




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






KATERINI
CS146
TCAGCAACTTATGGGAAGGTTCTGACT
420
TCAGCAACTTATGGGAAGGTTCTGACT
452




TTGGATGGAATGGTGGATTTCCATACA

TTGGATGGAgcaattcaacatacagag





CTGAAATGATTGTTCATCTA

aATGGTGGATTTCCATACACTGAAATG







ATTGTTCATCTA






KATERINI
CS147
TCAGCAACTTATGGGAAGGTTCTGACT
421
TCAGCAACTTATGGGAAGGTTCTGACT
453




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






KATERINI
CS150
TCAGCAACTTATGGGAAGGTTCTGACT
422
TCAGCAACTTATGGGAAGGTTCTGACT
454




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAACatACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






KATERINI
CS151
TCAGCAACTTATGGGAAGGTTCTGACT
423
TCAGCAACTTATGGGAAGGTTCTGACT
455




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAACatACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






KATERINI
CS148
TCAGCAACTTATGGGAAGGTTCTGACT
424
TCAGCAACTTATGGGAAGGTTCTGACT
456




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






KATERINI
CS149
TCAGCAACTTATGGGAAGGTTCTGACT
425
TCAGCAACTTATGGGAAGGTTCTGACT
457




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






KATERINI
CS152
TCAGCAACTTATGGGAAGGTTCTGACT
426
TCAGCAACTTATGGGAAGGTTCTGACT
458




TTGGATGGAGCAATTCAACATAGAGAA

TTGGATGGAGCAATTCAACATacAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






KATERINI
CS153
TCAGCAACTTATGGGAAGGTTCTGACT
427
TCAGCAACTTATGGGAAGGTTCTGACT
459




TTGGATGGAGCAATTCAGAGAATGGTG

TTGGATGGAGCAATTCAacatacaGAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTA

ATTGTTCATCTA






KATERINI
CS102
TCAGCAACTTATGGGAAGGTTCTGACT
428
TCAGCAACTTATGGGAAGGTTCTGACT
460




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






KATERINI
CS103
TCAGCAACTTATGGGAAGGTTCTGACT
429
TCAGCAACTTATGGGAAGGTTCTGACT
461




TTGGATGGAGCAATTCAACATAGAGAA

TTGGATGGAGCAATTCAACATacAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






TN90
CS143
TCAGCAACTTATGGGAAGGTTCTGACT
430
TCAGCAACTTATGGGAAGGTTCTGACT
462




TTGGATGGAGCAATTCAACAAATGGTG

TTGGATGGAGCAATTCAACAtacagag





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTA

ATTGTTCATCTA






TN90
18GH2169
TCAGCAACTTATGGGAAGGTTCTGACT
431
TCAGCAACTTATGGGAAGGTTCTGACT
463




TTGGATGGAGCAATTCAACATAGAGAA

TTGGATGGAGCAATTCAACATacAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






TN90
CS120
TCAGCAACTTATGGGAAGGTTCTGACT
432
TCAGCAACTTATGGGAAGGTTCTGACT
464




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
17GH1698-22
TCAGCAACTTATGGGAAGGTTCTGACT
433
TCAGCAACTTATGGGAAGGTTCTGACT
465




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
17GH1700-13
TCAGCAACTTATGGGAAGGTTCTGACT
434
TCAGCAACTTATGGGAAGGTTCTGACT
466




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
17GH1702-17
TCAGCAACTTATGGGAAGGTTCTGACT
435
TCAGCAACTTATGGGAAGGTTCTGACT
467




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
18GH2171
TCAGCAACTTATGGGAAGGTTCTGACT
436
TCAGCAACTTATGGGAAGGTTCTGACT
468




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
CS165
TCAGCAACTTATGGGAAGGTTCTGACT
437
TCAGCAACTTATGGGAAGGTTCTGACT
469




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
CS118
TCAGCAACTTATGGGAAGGTTCTGACT
438
TCAGCAACTTATGGGAAGGTTCTGACT
470




TTGGATATACAGAGAATGGTGGATTTC

TTGGATggagcaattcaacATACAGAG





CATACACTGAAATGATTGTTCATCTA

AATGGTGGATTTCCATACACTGAAATG







ATTGTTCATCTA






TN90
CS113
TCAGCAACTTATGGGAAGGTTCTGACT
439
TCAGCAACTTATGGGAAGGTTCTGACT
471




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacatACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTA

ATTGTTCATCTA






TN90
17GH1737-24
TCAGCAACTTATGGGAAGGTTCTGACT
440
TCAGCAACTTATGGGAAGGTTCTGACT
472




TTGGATGGAGCAATTCAATACAGAGAA

TTGGATGGAGCAATTCAAcaTACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTA

ATTGTTCATCTA






IZMIR
18GH2254-7
TCAGCAACTTATGGGAAGGTTCTGACT
441
TCAGCAACTTATGGGAAGGTTCTGACT
473




TTGGATGGAGCAATTCAGAGAATGGTG

TTGGATGGAGCAATTCAacatacaGAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTA

ATTGTTCATCTA
















TABLE 12B







A list of exemplary mutant alleles obtained in the PMT1a 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: 7) denote which nucleotides are


deleted in the mutant allele.















Mutant

Reference





Allele

Allele SEQ


Genotype
Line
Mutant Allele Sequence
SEQ ID NO.
Reference Allele Sequence
ID NO.





BASMA
CS107
TCAGCAACTTATGGGAAGGTTCTGACT
474
TCAGCAACTTATGGGAAGGTTCTGAC
506




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






BASMA
CS106
TCAGCAACTTATGGGAAGGTTCTGACT
475
TCAGCAACTTATGGGAAGGTTCTGAC
507




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






K326
CS115
TCAGCAACTTATGGGAAGGTTCTGACT
476
TCAGCAACTTATGGGAAGGTTCTGAC
508




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






K326
18GH2162
TCAGCAACTTATGGGAAGGTTCTGACT
477
TCAGCAACTTATGGGAAGGTTCTGAC
509




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






K326
CS111
TCAGCAACTTATGGGAAGGTTCTGACT
478
TCAGCAACTTATGGGAAGGTTCTGAC
510




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






K326
CS112
TCAGCAACTTATGGGAAGGTTCTGACT
479
TCAGCAACTTATGGGAAGGTTCTGAC
511




TTGGATGGAGCAATTCAACATAGAGAA

TTTGGATGGAGCAATTCAACATacAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTA

ATGATTGTTCATCTA






K326
17GH1678-60
TCAGCAACTTATGGGAAGGTTCTGACT
480
TCAGCAACTTATGGGAAGGTTCTGAC
512




TTGGATGGAGCAATTCAACATAGAGAA

TTTGGATGGAGCAATTCAACATacAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTA

ATGATTGTTCATCTA






K326
CS131
TCAGCAACTTATGGGAAGGTTCTGACT
481
TCAGCAACTTATGGGAAGGTTCTGAC
513




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS164
TCAGCAACTTATGGGAAGGTTCTGACT
482
TCAGCAACTTATGGGAAGGTTCTGAC
514




TTGGATGGAGCAATTCAGAGAATGGTG

TTTGGATGGAGCAATTCAacatacaG





GATTTCCATACACTGAAATGATTGTTC

AGAATGGTGGATTTCCATACACTGAA





ATCTA

ATGATTGTTCATCTA






KATERINI
CS163
TCAGCAACTTATGGGAAGGTTCTGACT
483
TCAGCAACTTATGGGAAGGTTCTGAC
515




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAACatACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTA

ATGATTGTTCATCTA






KATERINI
CS146
TCAGCAACTTATGGGAAGGTTCTGACT
484
TCAGCAACTTATGGGAAGGTTCTGAC
516




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS147
TCAGCAACTTATGGGAAGGTTCTGACT
485
TCAGCAACTTATGGGAAGGTTCTGAC
517




TTGGATGGAGCAATTCAGAGAATGGTG

TTTGGATGGAGCAATTCAacatacaG





GATTTCCATACACTGAAATGATTGTTC

AGAATGGTGGATTTCCATACACTGAA





ATCTA

ATGATTGTTCATCTA






KATERINI
CS150
TCAGCAACTTATGGGAAGGTTCTGACT
486
TCAGCAACTTATGGGAAGGTTCTGAC
518




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS151
TCAGCAACTTATGGGAAGGTTCTGACT
487
TCAGCAACTTATGGGAAGGTTCTGAC
519




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS148
TCAGCAACTTATGGGAAGGTTCTGACT
488
TCAGCAACTTATGGGAAGGTTCTGAC
520




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS149
TCAGCAACTTATGGGAAGGTTCTGACT
489
TCAGCAACTTATGGGAAGGTTCTGAC
521




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS152
TCAGCAACTTATGGGAAGGTTCTGACT
490
TCAGCAACTTATGGGAAGGTTCTGAC
522




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






KATERINI
CS153
TCAGCAACTTATGGGAAGGTTCTGACT
491
TCAGCAACTTATGGGAAGGTTCTGAC
523




TTGGATGGAGCAATTCAATACAGAGAA

TTTGGATGGAGCAATTCAAcaTACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTA

ATGATTGTTCATCTA






KATERINI
CS102
TCAGCAACTTATGGGAAGGTTCTGACT
492
TCAGCAACTTATGGGAAGGTTCTGAC
524




TTGGATGGAGCAATTCACAGAGAATGG

TTTGGATGGAGCAATTCAacataCAG





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTA

ATGATTGTTCATCTA






KATERINI
CS103
TCAGCAACTTATGGGAAGGTTCTGACT
493
TCAGCAACTTATGGGAAGGTTCTGAC
525




TTGGATGGAGCAATTCAGAGAATGGTG

TTTGGATGGAGCAATTCAacatacaG





GATTTCCATACACTGAAATGATTGTTC

AGAATGGTGGATTTCCATACACTGAA





ATCTA

ATGATTGTTCATCTA






TN90
CS143
TCAGCAACTTATGGGAAGGTTCTGACT
494
TCAGCAACTTATGGGAAGGTTCTGAC
526




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
18GH2169
TCAGCAACTTATGGGAAGGTTCTGACT
495
TCAGCAACTTATGGGAAGGTTCTGAC
527




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
CS120
TCAGCAACTTATGGGAAGGTTCTGACT
496
TCAGCAACTTATGGGAAGGTTCTGAC
528




TTGGATGGAGCAATTCAACATAGAATG

TTTGGATGGAGCAATTCAACATacag





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
17GH1698-22
TCAGCAACTTATGGGAAGGTTCTGACT
497
TCAGCAACTTATGGGAAGGTTCTGAC
529




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
17GH1700-13
TCAGCAACTTATGGGAAGGTTCTGACT
498
TCAGCAACTTATGGGAAGGTTCTGAC
530




TTGGATGGAGCAATTCACAGAGAATGG

TTTGGATGGAGCAATTCAacataCAG





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTA

ATGATTGTTCATCTA






TN90
17GH1702-17
TCAGCAACTTATGGGAAGGTTCTGACT
499
TCAGCAACTTATGGGAAGGTTCTGAC
531




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
18GH2171
TCAGCAACTTATGGGAAGGTTCTGACT
500
TCAGCAACTTATGGGAAGGTTCTGAC
532




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
CS165
TCAGCAACTTATGGGAAGGTTCTGACT
501
TCAGCAACTTATGGGAAGGTTCTGAC
533




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
CS118
TCAGCAACTTATGGGAAGGTTCTGACT
502
TCAGCAACTTATGGGAAGGTTCTGAC
534




TTGGATGGAGCAATTAGAATGGTGGAT

TTTGGATGGAGCAATTcaacatacag





TTCCATACACTGAAATGATTGTTCATC

AGAATGGTGGATTTCCATACACTGAA





TA

ATGATTGTTCATCTA






TN90
CS113
TCAGCAACTTATGGGAAGGTTCTGACT
503
TCAGCAACTTATGGGAAGGTTCTGAC
535




TTGGATGGAGCAATTCAACATAGAATG

TTTGGATGGAGCAATTCAACATacag





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






TN90
17GH1737-24
TCAGCAACTTATGGGAAGGTTCTGACT
504
TCAGCAACTTATGGGAAGGTTCTGAC
536




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacatACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTA

ATGATTGTTCATCTA






IZMIR
18GH2254-7
TCAGCAACTTATGGGAAGGTTCTGACT
505
TCAGCAACTTATGGGAAGGTTCTGAC
537




TTGGATGGAGCAATTCAACACTGGTGG

TTTGGATGGAGCAATTCAAcatacag





ATTTCCATACACTGAAATGATTGTTCA

agACTGGTGGATTTCCATACACTGAA





TCTA

ATGATTGTTCATCTA
















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

Allele SEQ


Genotype
Line
Mutant Allele Sequence
SEQ ID NO.
Reference Allele Sequence
ID NO.





BASMA
CS107
TCAGCAACTTATGGGAAGGTTCTGACT
538
TCAGCAACTTATGGGAAGGTTCTGAC
570




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






BASMA
CS106
TCAGCAACTTATGGGAAGGTTCTGACT
539
TCAGCAACTTATGGGAAGGTTCTGAC
571




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






K326
CS115
TCAGCAACTTATGGGAAGGTTCTGACT
540
TCAGCAACTTATGGGAAGGTTCTGAC
572




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






K326
18GH2162
TCAGCAACTTATGGGAAGGTTCTGACT
541
TCAGCAACTTATGGGAAGGTTCTGAC
573




TTGGATGGAGCAATTCAAGAATGGTGG

TTTGGATGGAGCAATTCAacacacag





ATTTCCATACACTGAAATGATTGTTCA

AGAATGGTGGATTTCCATACACTGAA





TCTT

ATGATTGTTCATCTT






K326
CS111
TCAGCAACTTATGGGAAGGTTCTGACT
542
TCAGCAACTTATGGGAAGGTTCTGAC
574




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






K326
CS112
TCAGCAACTTATGGGAAGGTTCTGACT
543
TCAGCAACTTATGGGAAGGTTCTGAC
575




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






K326
17GH1678-60
TCAGCAACTTATGGGAAGGTTCTGACT
544
TCAGCAACTTATGGGAAGGTTCTGAC
576




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






K326
CS131
TCAGCAACTTATGGGAAGGTTCTGACT
545
TCAGCAACTTATGGGAAGGTTCTGAC
577




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






KATERINI
CS164
TCAGCAACTTATGGGAAGGTTCTGACT
546
TCAGCAACTTATGGGAAGGTTCTGAC
578




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS163
TCAGCAACTTATGGGAAGGTTCTGACT
547
TCAGCAACTTATGGGAAGGTTCTGAC
579




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS146
TCAGCAACTTATGGGAAGGTTCTGACT
548
TCAGCAACTTATGGGAAGGTTCTGAC
580




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS147
TCAGCAACTTATGGGAAGGTTCTGACT
549
TCAGCAACTTATGGGAAGGTTCTGAC
581




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS150
TCAGCAACTTATGGGAAGGTTCTGACT
550
TCAGCAACTTATGGGAAGGTTCTGAC
582




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS151
TCAGCAACTTATGGGAAGGTTCTGACT
551
TCAGCAACTTATGGGAAGGTTCTGAC
583




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS148
TCAGCAACTTATGGGAAGGTTCTGACT
552
TCAGCAACTTATGGGAAGGTTCTGAC
584




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS149
TCAGCAACTTATGGGAAGGTTCTGACT
553
TCAGCAACTTATGGGAAGGTTCTGAC
585




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS152
TCAGCAACTTATGGGAAGGTTCTGACT
554
TCAGCAACTTATGGGAAGGTTCTGAC
586




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS153
TCAGCAACTTATGGGAAGGTTCTGACT
555
TCAGCAACTTATGGGAAGGTTCTGAC
587




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS102
TCAGCAACTTATGGGAAGGTTCTGACT
556
TCAGCAACTTATGGGAAGGTTCTGAC
588




TTGGATGGAGCAATTCAAGAATGGTGG

TTTGGATGGAGCAATTCAacacacag





ATTTCCATACACTGAAATGATTGTTCA

AGAATGGTGGATTTCCATACACTGAA





TCTT

ATGATTGTTCATCTT






KATERINI
CS103
TCAGCAACTTATGGGAAGGTTCTGACT
557
TCAGCAACTTATGGGAAGGTTCTGAC
589




TTGGATGGAGCAATTCAAGAATGGTGG

TTTGGATGGAGCAATTCAacacacag





ATTTCCATACACTGAAATGATTGTTCA

AGAATGGTGGATTTCCATACACTGAA





TCTT

ATGATTGTTCATCTT






TN90
CS143
TCAGCAACTTATGGGAAGGTTCTGACT
558
TCAGCAACTTATGGGAAGGTTCTGAC
590




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
18GH2169
TCAGCAACTTATGGGAAGGTTCTGACT
559
TCAGCAACTTATGGGAAGGTTCTGAC
591




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
CS120
TCAGCAACTTATGGGAAGGTTCTGACT
560
TCAGCAACTTATGGGAAGGTTCTGAC
592




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
17GH1698-22
TCAGCAACTTATGGGAAGGTTCTGACT
561
TCAGCAACTTATGGGAAGGTTCTGAC
593




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
17GH1700-13
TCAGCAACTTATGGGAAGGTTCTGACT
562
TCAGCAACTTATGGGAAGGTTCTGAC
594




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
17GH1702-17
TCAGCAACTTATGGGAAGGTTCTGACT
563
TCAGCAACTTATGGGAAGGTTCTGAC
595




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
18GH2171
TCAGCAACTTATGGGAAGGTTCTGACT
564
TCAGCAACTTATGGGAAGGTTCTGAC
596




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
CS165
TCAGCAACTTATGGGAAGGTTCTGACT
565
TCAGCAACTTATGGGAAGGTTCTGAC
597




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
CS118
TCAGCAACTTATGGGAAGGTTCTGACT
566
TCAGCAACTTATGGGAAGGTTCTGAC
598




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
CS113
TCAGCAACTTATGGGAAGGTTCTGACT
567
TCAGCAACTTATGGGAAGGTTCTGAC
599




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
17GH1737-24
TCAGCAACTTATGGGAAGGTTCTGACT
568
TCAGCAACTTATGGGAAGGTTCTGAC
600




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






IZMIR
18GH2254-7
TCAGCAACTTATGGGAAGGTTCTGACT
569
TCAGCAACTTATGGGAAGGTTCTGAC
601




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT
















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., 4nucleotides 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

Allele SEQ


Genotype
Line
Mutant Allele Sequence
SEQ ID NO.
Reference Allele Sequence
ID NO.





BASMA
CS107
TCAGCAACATATGGGAAGGTTCTGACT
602
TCAGCAACATATGGGAAGGTTCTGACT
634




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






BASMA
CS106
TCAGCAACATATGGGAAGGTTCTGACT
603
TCAGCAACATATGGGAAGGTTCTGACT
635




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






K326
CS115
TCAGCAACATATGGGAAGGTTCTGACT
604
TCAGCAACATATGGGAAGGTTCTGACT
636




TTGGATGGAGCAATTCAAAGAGAATGG

TTGGATGGAGCAATTCAACacacAGAG





TGGATTTCCATACACTGAAATGATTGT

AATGGTGGATTTCCATACACTGAAATG





TCATCTT

ATTGTTCATCTT






K326
18GH2162
TCAGCAACATATGGGAAGGTTCTGACT
605
TCAGCAACATATGGGAAGGTTCTGACT
637




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






K326
CS111
TCAGCAACATATGGGAAGGTTCTGACT
606
TCAGCAACATATGGGAAGGTTCTGACT
638




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






K326
CS112
TCAGCAACATATGGGAAGGTTCTGACT
607
TCAGCAACATATGGGAAGGTTCTGACT
639




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






K326
17GH1678-60
TCAGCAACATATGGGAAGGTTCTGACT
608
TCAGCAACATATGGGAAGGTTCTGACT
640




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






K326
CS131
TCAGCAACATATGGGAAGGTTCTGACT
609
TCAGCAACATATGGGAAGGTTCTGACT
641




TTGGATGGAGCAATTCAGAATGGTGGA

TTGGATGGAGCAATTCaacacacagAG





TTTCCATACACTGAAATGATTGTTCAT

AATGGTGGATTTCCATACACTGAAATG





CTT

ATTGTTCATCTT






KATERINI
CS164
TCAGCAACATATGGGAAGGTTCTGACT
610
TCAGCAACATATGGGAAGGTTCTGACT
642




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






KATERINI
CS163
TCAGCAACATATGGGAAGGTTCTGACT
611
TCAGCAACATATGGGAAGGTTCTGACT
643




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






KATERINI
CS146
TCAGCAACATATGGGAAGGTTCTGACT
612
TCAGCAACATATGGGAAGGTTCTGACT
644




TTGGATGGAGCAATTCAGAGAATGGTG

TTGGATGGAGCAATTcaacacaCAGAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTT

ATTGTTCATCTT






KATERINI
CS147
TCAGCAACATATGGGAAGGTTCTGACT
613
TCAGCAACATATGGGAAGGTTCTGACT
645




TTGGATGGAGCAATTCAGAGAATGGTG

TTGGATGGAGCAATTcaacacaCAGAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTT

ATTGTTCATCTT






KATERINI
CS150
TCAGCAACATATGGGAAGGTTCTGACT
614
TCAGCAACATATGGGAAGGTTCTGACT
646




TTGGATGGAGCAATTCAAAGAATGGTG

TTGGATGGAGCAATTCAAcacacagAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTT

ATTGTTCATCTT






KATERINI
CS151
TCAGCAACATATGGGAAGGTTCTGACT
615
TCAGCAACATATGGGAAGGTTCTGACT
647




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






KATERINI
CS148
TCAGCAACATATGGGAAGGTTCTGACT
616
TCAGCAACATATGGGAAGGTTCTGACT
648




TTGGATGGAGCAATTCAAAGAATGGTG

TTGGATGGAGCAATTCAAcacacagAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTT

ATTGTTCATCTT






KATERINI
CS149
TCAGCAACATATGGGAAGGTTCTGACT
617
TCAGCAACATATGGGAAGGTTCTGACT
649




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






KATERINI
CS152
TCAGCAACATATGGGAAGGTTCTGACT
618
TCAGCAACATATGGGAAGGTTCTGACT
650




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






KATERINI
CS153
TCAGCAACATATGGGAAGGTTCTGACT
619
TCAGCAACATATGGGAAGGTTCTGACT
651




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






KATERINI
CS102
TCAGCAACATATGGGAAGGTTCTGACT
620
TCAGCAACATATGGGAAGGTTCTGACT
652




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






KATERINI
CS103
TCAGCAACATATGGGAAGGTTCTGACT
621
TCAGCAACATATGGGAAGGTTCTGACT
653




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
CS143
TCAGCAACATATGGGAAGGTTCTGACT
622
TCAGCAACATATGGGAAGGTTCTGACT
654




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
18GH2169
TCAGCAACATATGGGAAGGTTCTGACT
623
TCAGCAACATATGGGAAGGTTCTGACT
655




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






TN90
CS120
TCAGCAACATATGGGAAGGTTCTGACT
624
TCAGCAACATATGGGAAGGTTCTGACT
656




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
17GH1698-22
TCAGCAACATATGGGAAGGTTCTGACT
625
TCAGCAACATATGGGAAGGTTCTGACT
657




TTGGATGGAGCAATTCAACACAGAATG

TTGGATGGAGCAATTCAACACacagAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






TN90
17GH1700-13
TCAGCAACATATGGGAAGGTTCTGACT
626
TCAGCAACATATGGGAAGGTTCTGACT
658




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
17GH1702-17
TCAGCAACATATGGGAAGGTTCTGACT
627
TCAGCAACATATGGGAAGGTTCTGACT
659




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
18GH2171
TCAGCAACATATGGGAAGGTTCTGACT
628
TCAGCAACATATGGGAAGGTTCTGACT
660




TTGGATGGAGCAATTCAGAGAATGGTG

TTGGATGGAGCAATTcaacacaCAGAG





GATTTCCATACACTGAAATGATTGTTC

AATGGTGGATTTCCATACACTGAAATG





ATCTT

ATTGTTCATCTT






TN90
CS165
TCAGCAACATATGGGAAGGTTCTGACT
629
TCAGCAACATATGGGAAGGTTCTGACT
661




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT






TN90
CS118
TCAGCAACATATGGGAAGGTTCTGACT
630
TCAGCAACATATGGGAAGGTTCTGACT
662




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
CS113
TCAGCAACATATGGGAAGGTTCTGACT
631
TCAGCAACATATGGGAAGGTTCTGACT
663




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






TN90
17GH1737-24
TCAGCAACATATGGGAAGGTTCTGACT
632
TCAGCAACATATGGGAAGGTTCTGACT
664




TTGGATGGAGCAATTCAACACAGAGAA

TTGGATGGAGCAATTCAacACACAGAG





TGGTGGATTTCCATACACTGAAATGAT

AATGGTGGATTTCCATACACTGAAATG





TGTTCATCTT

ATTGTTCATCTT






IZMIR
18GH2254-7
TCAGCAACATATGGGAAGGTTCTGACT
633
TCAGCAACATATGGGAAGGTTCTGACT
665




TTGGATGGAGCAATTCAACAGAGAATG

TTGGATGGAGCAATTCAacacACAGAG





GTGGATTTCCATACACTGAAATGATTG

AATGGTGGATTTCCATACACTGAAATG





TTCATCTT

ATTGTTCATCTT
















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 oneach 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

Allele SEQ


Genotype
Line
Mutant Allele Sequence
SEQ ID NO.
Reference Allele Sequence
ID NO.





BASMA
CS107
TCAGCAACATATGGGAAGGTTTTGACT
666
TCAGCAACATATGGGAAGGTTTTGAC
698




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






BASMA
CS106
TCAGCAACATATGGGAAGGTTTTGACT
667
TCAGCAACATATGGGAAGGTTTTGAC
699




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






K326
CS115
TCAGCAACATATGGGAAGGTTTTGACT
668
TCAGCAACATATGGGAAGGTTTTGAC
700




TTGGATGGAGCAATTCAACGAGAATGG

TTTGGATGGAGCAATTCAACacacaG





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTT

ATGATTGTTCATCTT






K326
18GH2162
TCAGCAACATATGGGAAGGTTTTGACT
669
TCAGCAACATATGGGAAGGTTTTGAC
701




TTGGATGGAGCAATTCAACGAGAATGG

TTTGGATGGAGCAATTCAACacacaG





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTT

ATGATTGTTCATCTT






K326
CS111
TCAGCAACATATGGGAAGGTTTTGACT
670
TCAGCAACATATGGGAAGGTTTTGAC
702




TTGGATGGAGCAATTCAAAGAGAATGG

TTTGGATGGAGCAATTCAAcacacAG





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTT

ATGATTGTTCATCTT






K326
CS112
TCAGCAACATATGGGAAGGTTTTGACT
671
TCAGCAACATATGGGAAGGTTTTGAC
703




TTGGATGGAGCAATTCAAAGAGAATGG

TTTGGATGGAGCAATTCAAcacacAG





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTT

ATGATTGTTCATCTT






K326
17GH1678-60
TCAGCAACATATGGGAAGGTTTTGACT
672
TCAGCAACATATGGGAAGGTTTTGAC
704




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






K326
CS131
TCAGCAACATATGGGAAGGTTTTGACT
673
TCAGCAACATATGGGAAGGTTTTGAC
705




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






KATERINI
CS164
TCAGCAACATATGGGAAGGTTTTGACT
674
TCAGCAACATATGGGAAGGTTTTGAC
706




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS163
TCAGCAACATATGGGAAGGTTTTGACT
675
TCAGCAACATATGGGAAGGTTTTGAC
707




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS146
TCAGCAACATATGGGAAGGTTTTGACT
676
TCAGCAACATATGGGAAGGTTTTGAC
708




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS147
TCAGCAACATATGGGAAGGTTTTGACT
677
TCAGCAACATATGGGAAGGTTTTGAC
709




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS150
TCAGCAACATATGGGAAGGTTTTGACT
678
TCAGCAACATATGGGAAGGTTTTGAC
710




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS151
TCAGCAACATATGGGAAGGTTTTGACT
679
TCAGCAACATATGGGAAGGTTTTGAC
711




TTGGATGGAGCAATTAGAATGGTGGAT

TTTGGATGGAGCAATTcaacacacag





TTCCATACACTGAAATGATTGTTCATC

AGAATGGTGGATTTCCATACACTGAA





TT

ATGATTGTTCATCTT






KATERINI
CS148
TCAGCAACATATGGGAAGGTTTTGACT
680
TCAGCAACATATGGGAAGGTTTTGAC
712




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS149
TCAGCAACATATGGGAAGGTTTTGACT
681
TCAGCAACATATGGGAAGGTTTTGAC
713




TTGGATGGAGCAATTAGAATGGTGGAT

TTTGGATGGAGCAATTcaacacacag





TTCCATACACTGAAATGATTGTTCATC

AGAATGGTGGATTTCCATACACTGAA





TT

ATGATTGTTCATCTT






KATERINI
CS152
TCAGCAACATATGGGAAGGTTTTGACT
682
TCAGCAACATATGGGAAGGTTTTGAC
714




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






KATERINI
CS153
TCAGCAACATATGGGAAGGTTTTGACT
683
TCAGCAACATATGGGAAGGTTTTGAC
715




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






KATERINI
CS102
TCAGCAACATATGGGAAGGTTTTGACT
684
TCAGCAACATATGGGAAGGTTTTGAC
716




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






KATERINI
CS103
TCAGCAACATATGGGAAGGTTTTGACT
685
TCAGCAACATATGGGAAGGTTTTGAC
717




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
CS143
TCAGCAACATATGGGAAGGTTTTGACT
686
TCAGCAACATATGGGAAGGTTTTGAC
718




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
18GH2169
TCAGCAACATATGGGAAGGTTTTGACT
687
TCAGCAACATATGGGAAGGTTTTGAC
719




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
CS120
TCAGCAACATATGGGAAGGTTTTGACT
688
TCAGCAACATATGGGAAGGTTTTGAC
720




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
17GH1698-22
TCAGCAACATATGGGAAGGTTTTGACT
689
TCAGCAACATATGGGAAGGTTTTGAC
721




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
17GH1700-13
TCAGCAACATATGGGAAGGTTTTGACT
690
TCAGCAACATATGGGAAGGTTTTGAC
722




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
17GH1702-17
TCAGCAACATATGGGAAGGTTTTGACT
691
TCAGCAACATATGGGAAGGTTTTGAC
723




TTGGATGGAGCAATTCAACAAGAATGG

TTTGGATGGAGCAATTCAACAcacag





TGGATTTCCATACACTGAAATGATTGT

AGAATGGTGGATTTCCATACACTGAA





TCATCTT

ATGATTGTTCATCTT






TN90
18GH2171
TCAGCAACATATGGGAAGGTTTTGACT
692
TCAGCAACATATGGGAAGGTTTTGAC
724




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
CS165
TCAGCAACATATGGGAAGGTTTTGACT
693
TCAGCAACATATGGGAAGGTTTTGAC
725




TTGGATGGAGCAATTCAACAGAGAATG

TTTGGATGGAGCAATTCAacacACAG





GTGGATTTCCATACACTGAAATGATTG

AGAATGGTGGATTTCCATACACTGAA





TTCATCTT

ATGATTGTTCATCTT






TN90
CS118
TCAGCAACATATGGGAAGGTTTTGACT
694
TCAGCAACATATGGGAAGGTTTTGAC
726




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
CS113
TCAGCAACATATGGGAAGGTTTTGACT
695
TCAGCAACATATGGGAAGGTTTTGAC
727




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






TN90
17GH1737-24
TCAGCAACATATGGGAAGGTTTTGACT
696
TCAGCAACATATGGGAAGGTTTTGAC
728




TTGGATGGAGCAATTCAACACAGAGAA

TTTGGATGGAGCAATTCAacACACAG





TGGTGGATTTCCATACACTGAAATGAT

AGAATGGTGGATTTCCATACACTGAA





TGTTCATCTT

ATGATTGTTCATCTT






IZMIR
18GH2254-7
TCAGCAACATATGGGAAGGTTTTGACT
697
TCAGCAACATATGGGAAGGTTTTGAC
729




TTGGATGGAGCAATTCAAGAATGGTGG

TTTGGATGGAGCAATTCAacacacag





ATTTCCATACACTGAAATGATTGTTCA

AGAATGGTGGATTTCCATACACTGAA





TCTT

ATGATTGTTCATCTT









Example 8. Alkaloid Analysis of PMT Edited Lines

Homozygous genome edited tobacco lines from Example 7, along with control lines, are grown 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
CS116
1
0.029



LC

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
CS116
1
0.015



LC
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












Pollen source
F1 hybrid seed



Male Sterile 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.









Percentage of Mold











Mold Rating
Significant















Variety
Replicate 1
Replicate 2
Replicate 3
Replicate 4
Mold
Some Mold
Little/No 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 (PMT RNAi)
G
G
G
G
G
G
G
G
G
G
G
G
0%
0%
100%


TN90 (PR50 RNai)
G
G
G
G
G
G
G
G
G
G
G
G
0%
0%
100%


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.





Claims
  • 1. A tobacco plant, or part thereof, comprising: (a) a first knockout mutant allele in a putrescine N-methyltransferase (PMT)1a gene (PMT1a), wherein a wildtype allele of the PMT1a gene encodes the polypeptide of SEQ ID NO: 12, and wherein the first knockout mutant allele in the PMT1a gene comprises the sequence of SEQ ID NO: 432; and(b) a second knockout mutant allele in a PMT1b gene, wherein a wildtype allele of the PMT1b gene encodes the polypeptide of SEQ ID NO: 11; and(c) a third knockout mutant allele in a PMT2 gene, wherein a wildtype allele of the PMT2 gene encodes the polypeptide of SEQ ID NO: 13; and(d) a fourth knockout mutant allele in a PMT3 gene, wherein a wildtype allele of the PMT3 gene encodes the polypeptide of SEQ ID NO: 14; and(e) a fifth knockout mutant allele in a PMT4 gene, wherein a wildtype allele of the PMT4 gene encodes the polypeptide of SEQ ID NO: 15,
  • 2. The tobacco plant, or part thereof, of claim 1, wherein said tobacco plant produces a leaf comprising a nicotine level less than 90% of the nicotine level of a leaf from a control tobacco plant not having said one or more mutant alleles when grown and processed under comparable conditions.
  • 3. The tobacco plant, or part thereof, of claim 1, wherein said tobacco plant produces a leaf comprising a total alkaloid level less than 90% of the total alkaloid level of a leaf from said control tobacco plant when grown and processed under comparable conditions.
  • 4. The tobacco plant, or part thereof, of claim 1, wherein one or more mutant alleles comprise a mutation in a sequence region selected from the group consisting of a promoter, 5′ untranslated region (UTR), first exon, first intron, second exon, second intron, third exon, 3′ UTR, terminator, and any combination thereof.
  • 5. The tobacco plant, or part thereof, of claim 1, wherein one or more mutant alleles comprise 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.
  • 6. The tobacco plant, or part thereof, of claim 1, wherein one or more mutant alleles result 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.
  • 7. The tobacco plant, or part thereof, of claim 1, wherein one or more mutant alleles comprise 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.
  • 8. The tobacco plant, or part thereof, of claim 1, wherein said tobacco plant produces a leaf comprising a nicotine level of less than 0.15% dry weight.
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