Light yellow locus in tobacco and application thereof

Abstract

To provide tobacco plants, tobacco seeds, compositions, and methods for producing populations of tobacco plants related to the identification and introgression of the light yellow locus in tobacco.SOLUTION: A method of producing a population of tobacco plants exhibiting a light yellow (PY) phenotype, the method comprising (a) genotyping a first population of tobacco plants or tobacco seeds for the presence of a marker locus associated with a PY Quantitative Trait Locus (QTL); (b) selecting the tobacco plants or tobacco seeds genotyped in step (a); and (c) producing a second population of tobacco plants or tobacco seeds comprising the PYQTL from the tobacco plants or tobacco seeds selected in step (b). Wherein the population comprises at least one tobacco plant or seed exhibiting a light yellow color phenotype.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 913,313, filed October 10, 2019; and U.S. Provisional Patent Application No. 62 / 913,414, filed October 10, 2019, both of which are incorporated by reference in their entireties. The Sequence Listing, which is 102,745 bytes (measured in MS-Windows) and contained in the file "P34737WO00_SL.txt", created October 9, 2020, has been filed electronically hereby and is incorporated by reference in its entirety.

[0002] Field This disclosure relates to tobacco plants, tobacco seeds, compositions, and methods related to the identification and introgression of the Pale Yellow (PY) locus in tobacco. This disclosure also relates to the generation of novel mutations within the PY locus in tobacco.

[0003] array A listing of the nucleic acid and amino acid sequences is provided in Table 1.

[0004] Table 1: Nucleic acid and amino acid sequences TIFF2026035667000002.tif78156TIFF2026035667000003.tif228156TIFF2026035667000004.tif228156TIFF2026035667000005.tif231156TIFF2026035667000006.tif231156TIFF2026035667000007.tif231156TIFF2026035667000008.tif231156TIFF2026035667000009.tif231156TIFF2026035667000010.tif231156TIFF2026035667000011.tif231156TIFF2026035667000012.tif231156TIFF2026035667000013.tif231156TIFF2026035667000014.tif231156TIFF2026035667000015.tif232156TIFF2026035667000016.tif227156TIFF2026035667000017.tif231156TIFF2026035667000018.tif223156TIFF2026035667000019.tif230156TIFF2026035667000020.tif232156TIFF2026035667000021.tif232156TIFF2026035667000022.tif231156TIFF2026035667000023.tif231156TIFF2026035667000024.tif231156TIFF2026035667000025.tif227156TIFF2026035667000026.tif232156TIFF2026035667000027.tif231156TIFF2026035667000028.tif205156

Background Art

[0005] Background In commercial tobacco (Nicotiana tabacum) varieties, accelerated senescence serves to reduce the levels of tobacco-specific nitrosamines (TSNAs) that would otherwise accumulate during the yellowing process associated with conventional varieties. Two loci, Yb1 and Yb2, are involved in controlling the rate of senescence in burley tobacco. A third locus, the pale yellow (PY) locus, is also known to be involved in controlling the rate of senescence in burley, flue-cured, and dark cultivars. The presence of the PY locus is also known to reduce TSNA levels.

[0006] The location and identity of the PY locus remain unknown, and incorporating the PY locus into desired tobacco varieties requires a time-consuming and subjective selection process. The present disclosure provides the location of the PY locus within the tobacco genome. The present disclosure also provides markers to facilitate accelerated breeding of the PY locus into different tobacco lines. Furthermore, incorporating the PY locus into low-alkaloid tobacco lines results in significant improvements in leaf quality as measured by the USDA Grade Index. Summary of the Invention

[0007] overview In one aspect, the disclosure provides a method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, the method comprising the steps of: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and linked within 20 centimorgans (cM) of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising a PY QTL and one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting a pale yellow phenotype.

[0008] In one aspect, the disclosure provides a method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, the method comprising: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and located within 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds from the one or more tobacco plants or seeds selected in step (b), the second population of tobacco plants or seeds comprising a PY QTL and one or more marker loci, wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting a pale yellow phenotype.

[0009] In one aspect, the disclosure provides a method for introgressing a pale yellow color (PY) QTL, the method comprising: (a) crossing a first tobacco plant comprising a PY quantitative trait locus (QTL) with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) selecting progeny plants or seeds produced in step (a) that comprise at least one PY-associated single nucleotide polymorphism (SNP) selected from the group consisting of: (i) a guanine at nucleotide 121 of SEQ ID NO: 1; (ii) a guanine at nucleotide 121 of SEQ ID NO: 2; (iii) a guanine at nucleotide 101 of SEQ ID NO: 3; (iv) a thymine at nucleotide 121 of SEQ ID NO: 4; or (v) a guanine at nucleotide 121 of SEQ ID NO: 5, wherein the selected progeny plants or seeds comprise a pale yellow color phenotype.

[0010] In one aspect, the disclosure provides a method of introgressing a pale yellow (PY) trait, the method comprising: (a) crossing a first tobacco plant that comprises a non-naturally occurring mutation in a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48 with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) selecting a progeny plant or seed produced in step (a) that comprises the non-naturally occurring mutation, wherein the progeny plant or seed comprises the PY trait.

[0011] In one aspect, the present disclosure provides a modified tobacco plant or portion thereof comprising a recombinant nucleic acid construct comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule, wherein the non-coding RNA molecule is capable of binding to RNA encoding an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50, wherein the non-coding RNA molecule suppresses expression of the amino acid sequence, and wherein the modified tobacco plant comprises a buff phenotype. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the expression of g58899 (SEQ ID NO: 24) measured using quantitative RT-PCR. The average RQ (e.g., fold change) of g58899 measured across three biological replicates for tobacco cultivars KY171, Pale Yellow KY171 (PY_KY171), and TI1372 at various time points after topping is shown. Actin was used as a control. [Figure 2] 1 shows the expression of g61524 (SEQ ID NO: 27) measured using quantitative RT-PCR. The average RQ (e.g., fold change) of g61524 measured across three biological replicates for tobacco cultivars KY171, Pale Yellow KY171 (PY_KY171), and TI1372 at various time points after topping is shown. Actin was used as a control. [Figure 3] 1 shows the expression of g58917 (SEQ ID NO: 25) measured using quantitative RT-PCR. The average RQ (e.g., fold change) of g58917 measured across three biological replicates for tobacco cultivars KY171, Pale Yellow KY171 (PY_KY171), and TI1372 at various time points after topping is shown. Actin was used as a control. [Figure 4] 1 shows RNA expression of TO plants overexpressing g58899 (SEQ ID NO: 54). [Figure 5] Photographs of four plant lines overexpressing g58899 (SEQ ID NO: 54) are shown. Black arrows point to the pale yellow tissue. [Figure 6] Photographs of plants expressing an RNAi construct (SEQ ID NO: 52) designed to silence g58899 (SEQ ID NO: 44) expression in tissue culture are shown. Black arrows point to pale yellow tissue. [Figure 7]1 shows RNA expression from TO plants expressing an RNAi construct (SEQ ID NO: 52) designed to silence g58899 (SEQ ID NO: 44) expression. [Figure 8] Photographs of TO plants expressing an RNAi construct (SEQ ID NO: 52) designed to suppress g58899 (SEQ ID NO: 44) expression are shown. The symbol "+" indicates wild-type-like expression. The symbols "(-)," "-," "--," and "---" refer to increasing levels of downregulation as determined by semiquantitative PCR, with (-) representing the least downregulation and -- representing the most downregulation within the group. WT refers to Narrow Leaf Madole control plants lacking the RNAi construct. g58905 refers to plants expressing an RNAi construct designed to suppress g58905 (SEQ ID NO: 26). Plants were imaged 3 weeks after topping unless otherwise noted. [Figure 9] Expression of g58899 (SEQ ID NO: 24) measured using quantitative RT-PCR is shown. Average RQ is shown for tobacco cultivar Narrow Leaf Madole (NLM; lacking the pale yellow trait), ds1771 (an F2 population generated by crossing NLM with TI1372), wild type (WT; lacking the pale yellow phenotype), ds1771 (HT) heterozygous for the pale yellow trait, ds1771 (HM) homozygous for the pale yellow trait, and TI1372 (source of the pale yellow trait) before topping (UT) and 24 hours after topping (24 hr). [Figure 10]Expression of g58917 (SEQ ID NO: 25) measured using quantitative RT-PCR is shown. Average RQ is shown for tobacco cultivar Narrow Leaf Madole (NLM; lacking the pale yellow trait), ds1771 (an F2 population generated by crossing NLM with TI1372), wild type (WT; lacking the pale yellow phenotype), ds1771 (HT) heterozygous for the pale yellow trait, ds1771 (HM) homozygous for the pale yellow trait, and TI1372 (source of the pale yellow trait) before topping (UT) and 24 hours after topping (24 hr). [Figure 11] Expression of g61524 (SEQ ID NO: 27) measured using quantitative RT-PCR is shown. Average RQ is shown for tobacco cultivar Narrow Leaf Madole (NLM; lacking the pale yellow trait), ds1771 (an F2 population generated by crossing NLM with TI1372), wild type (WT; lacking the pale yellow phenotype), ds1771 (HT) heterozygous for the pale yellow trait, ds1771 (HM) homozygous for the pale yellow trait, and TI1372 (source of the pale yellow trait) before topping (UT) and 24 hours after topping (24 hr). [Figure 12-1] 1 shows an alignment of the g58899 amino acid sequence (SEQ ID NO: 30) with the Arabidopsis amino acid sequences of BCM1 (SEQ ID NO: 55) and BCM2 (SEQ ID NO: 56). [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13-1] An alignment of g58899 amino acid sequences from tobacco varieties Narrow Leaf Madole (NLM; SEQ ID NO: 46), TN90 (SEQ ID NO: 30), and K326 (SEQ ID NO: 57) is shown. The g58899 consensus sequence is also provided as SEQ ID NO: 58. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14A]The relative quantification (RQ) of g58917_v2 (SEQ ID NO: 25) is shown. For each of Figures 14A, 14B, and 14C, tobacco lines K326, Pale Yellow (PY) K326, KY171 LC, PY KY 171 LC, TN90 LC, and PY TN90 LC were sampled. Samples were taken before topping (non-topping) and 24 hours after topping ("24 hours"). [Figure 14B] The RQ of g61524 (SEQ ID NO: 27) is shown. For each of Figures 14A, 14B, and 14C, tobacco lines K326, Pale Yellow (PY) K326, KY171 LC, PY KY 171 LC, TN90 LC, and PY TN90 LC were sampled. Samples were taken before topping (non-topping) and 24 hours after topping ("24 hours"). [Figure 14C] The RQ of g58899 (SEQ ID NO: 24) is shown. For each of Figures 14A, 14B, and 14C, tobacco lines K326, Pale Yellow (PY) K326, KY171 LC, PY KY 171 LC, TN90 LC, and PY TN90 LC were sampled. Samples were taken before topping ("No Topping") and 24 hours after topping ("24 hours"). DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of this disclosure described by the plural of that term. In the event of a conflict with terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or the "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York).

[0014] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety.

[0015] When a grouping of alternatives is presented, any and all combinations of the members comprising that grouping of alternatives are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventors specifically contemplate each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C, etc. The term "and / or," when used in a list of two or more items, refers to any one of the listed items by itself or in combination with any one or more of the other listed items. For example, the phrase "A and / or B" is intended to mean one or both of A and B, i.e., A alone, B alone, or A and B in combination. The phrase "A, B, and / or C" is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0016] When a range of numbers is provided herein, the range is understood to include not only the endpoints of the range, but also any number between the defined endpoints of the range. For example, "between 1 and 10" includes the numbers 1 and 10, as well as any number between 1 and 10.

[0017] When the term "about" is used, it is understood to mean plus or minus 10%. For example, "about 100" includes 90 to 110.

[0018] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the terms "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0019] The presence of the dominant pale yellow (PY) locus in tobacco plants accelerates leaf senescence. Accelerated senescence results in fewer tobacco-specific nitrosamines (TSNAs) accumulating in the leaves. Historically, the only way to introgress the PY locus into different tobacco lines or cultivars was by subjectively scoring the phenotype of plants suspected of carrying the PY locus, crossing the suspected plants to different tobacco plants, and then cultivating and screening the progeny. However, crossing may need to occur before the onset of PY-associated phenotypes. To address this, individual leaves can be treated with chemicals such as ethephon to increase the rate of senescence. This allows for phenotypic scoring earlier in the plant's lifespan than would otherwise be possible while waiting for senescence to occur naturally. These requirements have made the process of introgressing the PY locus time-consuming and labor-intensive.

[0020] The PY locus, when present, results in what is referred to as the "pale yellow (PY) phenotype" or "PY trait." The PY phenotype accelerates chlorophyll degradation and / or leaf maturation compared to control plants lacking the dominant PY locus. The PY phenotype is manifested by the early onset of leaf yellowing, ultimately resulting in completely yellow leaves. This yellowing occurs earlier than that seen in control tobacco plants of the same genetic background lacking the PY trait. Typically, the PY phenotype does not appear until after the tobacco plant is pinched.

[0021] In one aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least one day earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least two days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least three days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least four days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least five days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least six days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least seven days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after pinching at least eight days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant comprising the PY trait exhibits leaf yellowing of at least one green leaf after topping at least 9 days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant comprising the PY trait exhibits leaf yellowing of at least one green leaf after topping at least 10 days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions.In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after topping at least 14 days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after topping at least 18 days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions. In another aspect, a tobacco plant containing the PY trait exhibits leaf yellowing of at least one green leaf after topping at least 21 days earlier than a control tobacco plant of the same variety lacking the PY trait when grown under the same environmental conditions.

[0022] In tobacco, new leaves form as the stem grows, so the youngest leaves are the ones at the top of the stem and the oldest leaves are the ones at the bottom of the stem.

[0023] In one aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within one day of each other after the tobacco plant is topped. In another aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within two days of each other after the tobacco plant is topped. In another aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within three days of each other after the tobacco plant is topped. In another aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within four days of each other after the tobacco plant is topped. In another aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within five days of each other after the tobacco plant is topped. In another aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within six days of each other after the tobacco plant is topped. In another aspect, the youngest and oldest green leaves remaining on a tobacco plant containing the PY trait after topping begin to turn yellow within seven days of each other after the tobacco plant is topped.

[0024] It is understood in the art that the PY phenotype may manifest slightly differently depending on the tobacco variety. Because conventional dark tobacco varieties do not turn yellow during the aging process, the PY phenotype is most easily observed in dark tobacco varieties. Typically, the leaves of dark tobacco varieties continue to swell, thicken, and become more brittle after pinching, but the leaves remain dark green. Occasionally, leaves may develop spots that are not green in color. When conventional dark tobacco varieties are harvested, approximately 5 to 7 weeks after pinching, little yellowing of the leaves or stems is observed. In contrast, when the PY trait is introgressed into dark tobacco varieties, plants begin to exhibit leaf yellowing within two weeks of pinching. The plants exhibit yellowing of all leaves, especially the leaf blades (the midrib usually remains green). The plants also exhibit yellow stems.

[0025] In one aspect, a dark tobacco plant containing the PY trait exhibits yellowing of at least one green leaf after pinching.

[0026] Unlike dark tobacco varieties, traditional burley tobacco varieties yellow during the aging process. When burley tobacco is topped, some of the lower (older) leaves may already have begun to yellow. Traditional burley tobacco continues to yellow from bottom to top after topping. However, when burley tobacco has the PY trait, the plant yellows at an accelerated pace, with all leaves beginning to yellow simultaneously, often within one week of topping.

[0027] As used herein, "yellowing" refers to the loss of chlorophyll in tobacco leaf or stem tissue, resulting in a yellow coloration.

[0028] The present disclosure identifies, for the first time, the chromosomal location of the PY locus. The present disclosure also provides markers suitable for tracking the PY locus in tobacco.

[0029] In one aspect, the disclosure provides a method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, the method comprising: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and linked within 20 centimorgans of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising a PY QTL and one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting the PY phenotype.

[0030] In another aspect, the disclosure provides a method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, the method comprising the steps of: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and linked within 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising a PY QTL and one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting the PY phenotype.

[0031] marker As used herein, the phrase "associated with" or "linked to" refers to a recognizable and / or assayable relationship between two entities.For example, the phrase "associated with PY trait" or "associated with PY QTL" refers to a trait, locus, gene, allele, marker, phenotype, etc., or its expression, whose presence or absence can affect the degree, extent, and / or speed of a plant or its part of interest that has PY trait or PY QTL.In this way, if a marker is linked to a trait, and the presence of the marker indicates whether and / or to what extent a desired trait or trait form occurs in a plant / germplasm that contains the marker, then the marker is "associated with" the trait.Similarly, if a marker is linked to an allele, and the presence of the marker indicates whether the allele exists in a plant / germplasm that contains the marker, then the marker is "associated with" the allele. For example, a "marker associated with the PY trait" refers to a marker whose presence or absence can be used to predict whether and to what extent a plant will exhibit the PY phenotype.

[0032] In one aspect, the PY QTL is located on chromosome 15 of the tobacco genome between SSR markers PT51549 and PT55414.

[0033] As used herein, a "locus" refers to a fixed position on a chromosome. In one aspect, a locus comprises a gene. In another aspect, a locus comprises a marker. A locus can represent a single nucleotide, a few nucleotides, or multiple nucleotides within a genomic region. As used herein, a "marker," "molecular marker," or "marker locus" refers to a nucleic acid sequence that is sufficiently unique to characterize a specific locus on a genome. Any detectable polymorphic trait can be used as a marker as long as it is differentially inherited and shows linkage disequilibrium with the phenotypic trait of interest. When a trait is said to be linked to a given marker, it will be understood that the actual DNA segment of the sequence that affects the trait generally co-segregates with the marker. If markers are identified on both sides of the trait, more precise and clear localization of the trait can be obtained. A relatively simple molecular test can detect the presence of a trait by measuring the appearance of a marker in the progeny of a cross, without actually assessing the appearance of the trait itself, which can be difficult and time-consuming because actual evaluation of the trait requires growing the plant to a stage and / or under environmental conditions where the trait can be expressed. It is understood in the art that a marker can contain two or more alleles, and that two or more alleles can be associated with a given trait.

[0034] As used herein, " allele " refers to the alternative nucleic acid sequence at a specific locus.The length of allele can be as small as one nucleotide base.For example, the first allele can occur on one chromosome, and the second allele can occur on a second homologous chromosome, for example, for different chromosomes of heterozygous individuals, or between different homozygous or heterozygous individuals in a population.

[0035] Genetic linkage refers to the tendency of DNA loci (e.g., genes, markers) to be inherited together during the meiotic phase of sexual reproduction. Loci that are physically close to each other are more likely to be genetically "linked" to each other. Linkage can refer to either genetic linkage or physical linkage. Genetic linkage is usually measured using centimorgans, while physical linkage is usually measured in nucleotides.

[0036] As used herein, "centimorgan" (cM) refers to the distance between chromosomal locations (also called loci or markers) where the expected average number of intervening chromosomal crossovers in a single generation is 0.01. If the genetic distance between two loci is greater than 50 cM, the two loci are generally considered to be genetically unlinked.

[0037] In one aspect, one or more marker loci are within 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 17.5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 15 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 14 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 13 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 12 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 11 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 9 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 8 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 7 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 6 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 4 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, one or more marker loci are within 3 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 2 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.25 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

[0038] In one aspect, one or more marker loci are within 0.01 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.05 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.1 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.5 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 5 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10 cM to 20 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.01 cM to 15 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.01 cM to 10 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.01 cM to 7.5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.01 cM to 5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.01 cM to 2.5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, one or more marker loci are within 0.01 cM to 1 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.5 cM to 10 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 0.5 cM to 5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

[0039] In one aspect, one or more marker loci are within 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 75,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 40,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 30,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 25,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 17,500,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 15,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 12,500,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 9,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, one or more marker loci are within 8,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 7,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 6,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 5,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 4,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 3,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 2,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 750,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 500,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 250,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, one or more marker loci are within 75,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 50,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 25,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 5,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 2,500 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 750 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 500 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 250 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.

[0040] In one aspect, one or more marker loci are within 1 nucleotide to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10 nucleotides to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100 nucleotides to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000 nucleotides to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000 to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100,000 to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000,000 to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000,000 to 100,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1 nucleotide to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10 nucleotides to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, one or more marker loci are within 100 nucleotides to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000 nucleotides to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000 nucleotides to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100,000 nucleotides to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000,000 to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000,000 to 50,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1 to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10 to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100 nucleotides to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000 nucleotides to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, one or more marker loci are within 10,000 to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100,000 to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000,000 to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000,000 to 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1 nucleotide to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10 nucleotides to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100 nucleotides to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 1,000 nucleotides to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 10,000 to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, one or more marker loci are within 100,000 to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.In one aspect, the one or more marker loci are within 1,000,000 nucleotides to 10,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.

[0041] In one aspect, the locus comprises SEQ ID NO: 1. In another aspect, the locus comprises SEQ ID NO: 2. In another aspect, the locus comprises SEQ ID NO: 3. In another aspect, the locus comprises SEQ ID NO: 4. In another aspect, the locus comprises SEQ ID NO: 5.

[0042] Any type of polymorphic marker is contemplated for use in the methods and compositions provided herein. In one aspect, the marker locus is selected from the group consisting of SEQ ID NOs: 1-5. In one aspect, the marker locus comprises SEQ ID NO: 1. In one aspect, the marker locus comprises SEQ ID NO: 2. In one aspect, the marker locus comprises SEQ ID NO: 3. In one aspect, the marker locus comprises SEQ ID NO: 4. In one aspect, the marker locus comprises SEQ ID NO: 5.

[0043] In one aspect, the one or more marker loci comprise a single nucleotide polymorphism (SNP) selected from the group consisting of: (a) guanine at position 121 of SEQ ID NO: 1; (b) guanine at position 121 of SEQ ID NO: 2; (c) guanine at position 101 of SEQ ID NO: 3; (d) thymine at position 121 of SEQ ID NO: 4; and (e) guanine at position 121 of SEQ ID NO: 5.

[0044] In one aspect, the marker locus comprises a guanine at position 121 of SEQ ID NO: 1. In another aspect, the marker locus comprises a guanine at position 121 of SEQ ID NO: 2. In another aspect, the marker locus comprises a guanine at position 101 of SEQ ID NO: 3. In another aspect, the marker locus comprises a thymine at position 121 of SEQ ID NO: 4. In another aspect, the marker locus comprises a guanine at position 121 of SEQ ID NO: 5.

[0045] In one aspect, the plant or seed is homozygous for the SNP. In another aspect, the plant or seed is heterozygous for the SNP.

[0046] In one aspect, the plant or seed is homozygous for the marker locus. In another aspect, the plant or seed is heterozygous for the marker locus.

[0047] In one aspect, the marker loci include one or more single nucleotide polymorphism markers. In another aspect, the marker loci include one or more insertion-deletion (INDEL) markers. In another aspect, the marker loci include one or more simple sequence repeat (SSR) markers. In another aspect, the marker loci include one or more restriction fragment length polymorphism (RFLP) markers. In another aspect, the marker loci include one or more random amplified polymorphic DNA (RAPD) markers. In another aspect, the marker loci include one or more amplified fragment length polymorphism (AFLP) markers. In one aspect, the one or more marker loci are selected from the group consisting of one or more SNP markers, one or more INDEL markers, one or more SSR markers, one or more RFLP markers, one or more RAPD markers, and one or more AFLP markers.

[0048] It is understood that genotyping involves determining the genetic makeup of individual plants or plant cells by using molecular assays to examine DNA sequences and compare the sequences with reference sequences. Genotyping is different from visual phenotyping, which is performed solely by visual inspection of plants or plant cells. In one aspect, genotyping does not include visual phenotyping of plants or plant cells.

[0049] In one aspect, genotyping involves detecting one or more marker loci. In another aspect, genotyping involves detecting one or more alleles or one or more marker loci. Detecting the presence of a marker locus or a specific allele of a marker locus can include any suitable method or technique or method known in the art. Non-limiting examples for detecting markers or marker alleles include gel electrophoresis, DNA sequencing, RNA sequencing, Southern blot, and microarray technology.

[0050] In one aspect, genotyping involves the use of oligonucleotide probe.As used herein, "oligonucleotide probe" refers to an oligonucleotide (synthetic or naturally occurring) that is complementary (not necessarily completely complementary) to the polynucleotide of interest and forms a duplex structure by hybridization with at least one strand of the polynucleotide of interest.In one aspect, the oligonucleotide probe comprises DNA.In another aspect, the oligonucleotide probe comprises RNA.In another aspect, the oligonucleotide probe is single-stranded.In another aspect, the oligonucleotide probe is partially double-stranded.In one aspect, the oligonucleotide probe can function as a primer for PCR.

[0051] Typically, oligonucleotide probes comprise 10 to 50 nucleotides in length, although longer or shorter sequences can be utilized. In one aspect, the oligonucleotide probe comprises 15 to 40 nucleotides. In one aspect, the oligonucleotide probe comprises 15 to 35 nucleotides. In one aspect, the oligonucleotide probe comprises 15 to 30 nucleotides. In one aspect, the oligonucleotide probe comprises 20 to 40 nucleotides. In one aspect, the oligonucleotide probe comprises 20 to 35 nucleotides. In one aspect, the oligonucleotide probe comprises 10 to 100 nucleotides. In one aspect, the oligonucleotide probe comprises 10 to 75 nucleotides. In one aspect, the oligonucleotide probe comprises 10 to 50 nucleotides. In one aspect, the oligonucleotide probe comprises 10 to 40 nucleotides. In one aspect, the oligonucleotide probe comprises 10 to 30 nucleotides.

[0052] In one aspect, the oligonucleotide probe comprises at least 18 nucleotides. In one aspect, the oligonucleotide probe comprises at least 19 nucleotides. In one aspect, the oligonucleotide probe comprises at least 20 nucleotides. In one aspect, the oligonucleotide probe comprises at least 21 nucleotides. In one aspect, the oligonucleotide probe comprises at least 22 nucleotides. In one aspect, the oligonucleotide probe comprises at least 23 nucleotides. In one aspect, the oligonucleotide probe comprises at least 24 nucleotides. In one aspect, the oligonucleotide probe comprises at least 25 nucleotides. In one aspect, the oligonucleotide probe comprises at least 26 nucleotides. In one aspect, the oligonucleotide probe comprises at least 27 nucleotides. In one aspect, the oligonucleotide probe comprises at least 28 nucleotides. In one aspect, the oligonucleotide probe comprises at least 29 nucleotides. In one aspect, the oligonucleotide probe comprises at least 30 nucleotides. In one aspect, the oligonucleotide probe comprises at least 31 nucleotides. In one aspect, the oligonucleotide probe comprises at least 32 nucleotides. In one aspect, the oligonucleotide probe comprises at least 33 nucleotides. In one aspect, the oligonucleotide probe comprises at least 34 nucleotides. In one aspect, the oligonucleotide probe comprises at least 35 nucleotides.

[0053] The probe can further comprise a detectable label. The detectable label can be at the 5' end, the 3' end, or inside the oligonucleotide probe. The oligonucleotide probe can be designed to hybridize with a specific marker or a specific allele of a marker. Non-limiting examples of detectable labels include biotin, fluorophores (e.g., green fluorescent protein, Texas Red®, VIC™, JUN™, ABY™), and radioisotopes (e.g., phosphorus-32, sulfur-35, iodine-125).

[0054] In one aspect, the oligonucleotide probe is a TaqMan™ probe. TaqMan™ probes are often used to enhance the specificity of quantitative PCR. TaqMan™ probes rely on the 5' to 3' exonuclease activity of Taq polymerase to cleave the dual-labeled probe during hybridization with complementary target sequence and fluorophore-based detection. As with other quantitative PCR methods, the resulting fluorescent signal allows quantitative measurement of product accumulation during the exponential phase of PCR; however, TaqMan™ probes significantly increase the specificity of detection.

[0055] In one aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34 to 42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34 to 42. In another aspect, the oligonucleotide probe comprises a nucleic acid sequence 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34 to 42.

[0056] In one aspect, the oligonucleotide probe is at least 80% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 85% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 90% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 91% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 92% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 93% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 94% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 95% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 96% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 97% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.In another aspect, the oligonucleotide probe is at least 98% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 99% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is 100% identical to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0057] In one aspect, the oligonucleotide probe is at least 80% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 85% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 90% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 91% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 92% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 93% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 94% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 95% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 96% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 97% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.In another aspect, the oligonucleotide probe is at least 98% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is at least 99% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the oligonucleotide probe is 100% identical to at least 21 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0058] In one aspect, the oligonucleotide probe is adjacent to the polymorphic nucleotide position of one or more marker loci. As used herein, "adjacent" refers to the nearest end (3' or 5') of the oligonucleotide probe and a distance of 0 to 50 nucleotides from the polymorphic nucleotide position. As used herein, "polymorphic nucleotide position" refers to a difference (e.g., insertion, deletion, substitution) between two or more alleles of a given marker locus. The polymorphic nucleotide position can be found by making a pairwise comparison between allele sequences. For example, if a first allele contains the nucleotide sequence ATTTG and a second allele contains the nucleotide sequence TTTTG, the first allele will be the "polymorphic nucleotide position."

[0059] In one aspect, genotyping comprises detecting haplotype.As used herein, " haplotype " refers to a group of two or more loci inherited from one parent.Loci can include genes, markers, or a combination of genes and markers.Usually, the loci described by haplotypes are physically and genetically linked.Haplotype can also refer to the combination of SNPs located within a single locus.

[0060] In one aspect, detecting the haplotype includes detecting at least two single nucleotide polymorphisms (SNPs) selected from the group consisting of a guanine at nucleotide 121 of SEQ ID NO: 1, a guanine at nucleotide 121 of SEQ ID NO: 2, a guanine at nucleotide 101 of SEQ ID NO: 3, a thymine at nucleotide 121 of SEQ ID NO: 4, and a guanine at nucleotide 121 of SEQ ID NO: 5.

[0061] Gene transfer In one aspect, the disclosure provides a method for introgressing a pale yellow (PY) quantitative trait locus (QTL), the method comprising: (a) crossing a first tobacco plant containing a PY QTL with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) selecting progeny plants or seeds produced in step (a) that contain at least one PY-associated single nucleotide polymorphism selected from the group consisting of: (i) a guanine at nucleotide 121 of SEQ ID NO: 1; (ii) a guanine at nucleotide 121 of SEQ ID NO: 2; (iii) a guanine at nucleotide 101 of SEQ ID NO: 3; (iv) a thymine at nucleotide 121 of SEQ ID NO: 4, and (v) a guanine at nucleotide 121 of SEQ ID NO: 5, wherein the selected progeny plants or seeds comprise a PY phenotype.

[0062] As used herein, "introgression" refers to the transfer of a desired allele at a genetic locus from one genetic background (eg, genotype) to another genetic background.

[0063] As used herein, a "PY-associated single nucleotide polymorphism" refers to a polymorphism that segregates with a PY QTL.

[0064] As used herein, "genotype" refers to the genetic makeup of a plant or cell. Two tobacco plants from different tobacco lines or varieties will be understood to have different genotypes. Alternatively, inbred plants usually contain the same genotype. However, without limitation, if a PY QTL is introgressed into a single TN 90 plant, the single TN 90 plant containing the PY QTL will have a different genotype from all TN 90 plants lacking the PY QTL.

[0065] mutation In one aspect, the disclosure provides a method of introgressing a pale yellow (PY) trait, the method comprising: (a) crossing a first tobacco plant that comprises a non-naturally occurring mutation in a nucleic acid selected from the group consisting of SEQ ID NOs: 16-21 and 48 with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) selecting a progeny plant or seed produced in step (a) that comprises the non-naturally occurring mutation, wherein the progeny plant or seed comprises the PY trait.

[0066] In one aspect, the present disclosure provides a modified tobacco plant or portion thereof comprising a non-naturally occurring mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48, wherein the modified tobacco plant comprises a buff phenotype, and wherein the mutation is compared to a control tobacco plant of the same tobacco variety.

[0067] In another aspect, the present disclosure provides a modified tobacco plant or portion thereof comprising a non-naturally occurring mutation in a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50, wherein the modified tobacco plant comprises a buff phenotype, and wherein the mutation is compared to a control tobacco plant of the same tobacco variety.

[0068] In one aspect, the plant or seed is homozygous for a non-naturally occurring mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the plant or seed is heterozygous for a non-naturally occurring mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0069] In one aspect, the plant or seed is homozygous for a non-naturally occurring mutation in a nucleic acid sequence encoding an amino acid selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the plant or seed is heterozygous for a non-naturally occurring mutation in a nucleic acid sequence encoding an amino acid selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. As used herein, a "mutation" refers to an inherited genetic modification that is genetically introduced to alter the expression or activity of a product encoded by a gene. Such a modification can be in any sequence region of a gene, for example, the promoter, 5' UTR, exons, introns, 3' UTR, or terminator region. In one aspect, the mutation reduces, inhibits, or eliminates expression or activity of the gene product. In another aspect, the mutation increases, elevates, strengthens, or enhances expression or activity of the gene product.

[0070] In one aspect, mutation is " non-natural " or " non-naturally occurring " mutation.As used herein, " non-natural " or " non-naturally occurring " mutation refers to the non-naturally occurring mutation that occurs by human intervention, and does not correspond to the naturally occurring mutation that occurs without human intervention.Non-limiting examples of human intervention include mutagenesis (for example, chemical mutagenesis, ionizing radiation mutagenesis) and targeted gene modification (for example, CRISPR-based method, TALEN-based method, zinc finger-based method).Non-naturally occurring mutation and non-naturally occurring mutation do not include the naturally occurring mutation that occurs naturally (for example, due to abnormal DNA replication in plant germ lineage).

[0071] It should be understood that when identifying mutations, the reference DNA sequence should be derived from the same tobacco variety.For example, if the modified tobacco plant containing mutations is derived from variety TN90, the endogenous reference sequence should be the endogenous TN90 sequence, not the homologous sequence from a different tobacco variety (e.g., K326).Similarly, if the modified tobacco cell containing mutations is TN90 cell, the endogenous reference sequence should be the endogenous TN90 sequence, not the homologous sequence from a tobacco cell from a different tobacco variety (e.g., K326).

[0072] In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the endogenous nucleic acid sequence comprising a non-naturally occurring mutation comprises a nucleic acid sequence 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0073] In one aspect, the mutation provided herein creates a dominant allele at the mutated locus. A dominant allele is an allele that masks the contribution of a second allele at the same locus. A dominant allele can be a "dominant negative allele" or a "dominant positive allele." A dominant negative allele, or an inhibitory allele, is an allele that acts against the function of a normal allele. A dominant negative allele usually does not function normally, either directly inhibiting the activity of wild-type protein (for example, by dimerization) or inhibiting the activity of a second protein (for example, an activator or downstream component of a pathway) that is required for the normal function of wild-type protein. For example, a dominant negative allele disables or reduces the normal function of an allele in a heterozygous or homozygous state. A dominant positive allele can increase normal gene function (for example, hypermorphism) or provide a new function to a gene (for example, neomorphism). A semi-dominant allele occurs when the penetrance of the associated phenotype in individuals heterozygous for the allele is less than the penetrance observed in individuals homozygous for the allele.

[0074] In one aspect, the mutations provided herein create a dominant negative allele at the mutated locus. In another aspect, the mutations provided herein create a dominant positive allele at the mutated locus.

[0075] As used herein, "inducing" a mutation refers to creating a mutation in a polynucleotide sequence through human intervention. Many suitable methods for inducing mutations in tobacco are known in the art. Non-limiting examples of such methods include the use of chemical mutagens, irradiation, and nucleases. In one aspect, inducing a mutation includes the use of an agent selected from the group consisting of chemical mutagens, irradiation, transposons, Agrobacterium, and nucleases.

[0076] In one aspect, inducing mutations comprises the use of a chemical mutagen. In one aspect, the chemical mutagen comprises ethyl methanesulfonate (EMS).

[0077] In another aspect, inducing mutations involves the use of irradiation. In one aspect, irradiation involves gamma rays, X-rays, or ionizing radiation. In another aspect, irradiation involves the use of fast neutrons.

[0078] In one aspect, the mutagenesis involves the use of a transposon, hi another aspect, the mutagenesis involves the use of Agrobacterium.

[0079] In a further aspect, inducing mutations involves the use of a nuclease. In one aspect, the nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR / Cas9 nucleases, CRISPR / Cpf1 nucleases, CRISPR / CasX nucleases, CRISPR / CasY nucleases, and Csm1 nucleases. In one aspect, inducing mutations involves the use of CRISPR / Cas9 nucleases. In one aspect, inducing mutations involves the use of CRISPR / Cpf1 nucleases. In one aspect, inducing mutations involves the use of CRISPR / CasX nucleases. In one aspect, inducing mutations involves the use of CRISPR / CasY nucleases. In one aspect, the mutagenesis comprises the use of Csm1 nuclease.

[0080] Several types of mutations are known in the art. In one aspect, a mutation includes an insertion. "Insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation includes a deletion. "Deletion" refers to the removal of one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation includes a substitution. "Substitution" refers to the replacement of one or more nucleotides or amino acids from a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation includes an inversion. "Inversion" refers to when a segment of a polynucleotide or amino acid sequence is inverted end-to-end. In one aspect, the mutations provided herein include a mutation selected from the group consisting of an insertion, a deletion, a substitution, and an inversion.

[0081] In one aspect, the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the wild-type nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0082] In one aspect, the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to the wild-type nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.

[0083] In one aspect, the non-natural mutation comprises one or more mutation types selected from the group consisting of nonsense mutation, missense mutation, frameshift mutation, splice site mutation, and any combination thereof. As used herein, "nonsense mutation" refers to a mutation in a nucleic acid sequence that introduces a premature stop codon into an amino acid sequence by the nucleic acid sequence. As used herein, "missense mutation" refers to a mutation in a nucleic acid sequence that causes a substitution in the amino acid sequence encoded by the nucleic acid sequence. As used herein, "frameshift mutation" refers to an insertion or deletion in a nucleic acid sequence that shifts the frame for translating a nucleic acid sequence into an amino acid sequence. "Splice site mutation" refers to a mutation in a nucleic acid sequence that retains an intron for protein translation or so that an exon is excluded from protein translation. Splice site mutations can cause nonsense mutations, missense mutations, or frameshift mutations.

[0084] Mutations in the coding region of a gene (e.g., exon mutations) can result in a truncated protein or polypeptide when the mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In one aspect, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide contains at least one fewer amino acid than an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, a truncated form of protein A can contain 1 to 99 amino acids.

[0085] Without being limited by any scientific theory, one way to cause the truncation of a protein or polypeptide is by introducing a premature stop codon in the mRNA transcript of an endogenous gene. In one aspect, the present disclosure provides a mutation that results in a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet in an mRNA transcript that signals the termination of protein translation. A "premature stop codon" refers to a stop codon that is located earlier (e.g., 5') than the normal stop codon position in an endogenous mRNA transcript. Several stop codons are known in the art, including, but not limited to, "UAG", "UAA", "UGA", "TAG", "TAA", and "TGA".

[0086] In one aspect, the mutation provided herein comprises a null mutation.As used herein, " null mutation" refers to the mutation that causes the protein encoded by the gene that contains the mutation to lose all function, or the mutation that causes the small RNA encoded by the genomic locus to lose all function.Non-expressing mutation can cause the lack of mRNA transcript production, the lack of small RNA transcript production, the lack of protein function, or a combination thereof.

[0087] The mutations provided herein can be located in any part of endogenous gene. In one aspect, the mutations provided herein are located in the exons of endogenous gene. In another aspect, the mutations provided herein are located in the introns of endogenous gene. In a further aspect, the mutations provided herein are located in the 5' untranslated region (UTR) of endogenous gene. In yet another aspect, the mutations provided herein are located in the 3'-UTR of endogenous gene. In yet another aspect, the mutations provided herein are located in the promoter of endogenous gene. In yet another aspect, the mutations provided herein are located in the terminator of endogenous gene.

[0088] Screening and selection of mutagenized tobacco plants can be performed by any methodology known to those skilled in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detecting polynucleotides, Northern blot, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, next-generation sequencing technology (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blot, immunoprecipitation, and enzyme immunoassay for detecting polypeptides. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining, can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the referenced techniques are known.

[0089] In one aspect, the mutation of the endogenous gene results in a reduced level of expression compared to the endogenous gene lacking the mutation. In another aspect, the mutation of the endogenous gene results in an increased level of expression compared to the endogenous gene lacking the mutation.

[0090] In one aspect, the non-naturally occurring mutation results in a reduced level of expression compared to expression of the gene in a control tobacco plant. In one aspect, the non-naturally occurring mutation results in an increased level of expression compared to expression of the gene in a control tobacco plant.

[0091] In a further aspect, the mutation of the endogenous gene results in a reduced level of activity by the protein or polypeptide encoded by the endogenous gene having the mutation compared to the protein or polypeptide encoded by the endogenous gene lacking the mutation, hi a further aspect, the mutation of the endogenous gene results in an increased level of activity by the protein or polypeptide encoded by the endogenous gene having the mutation compared to the protein or polypeptide encoded by the endogenous gene lacking the mutation.

[0092] In one aspect, the non-naturally occurring mutation results in a reduced level of activity by a protein or polypeptide encoded by a polynucleotide comprising the non-naturally occurring mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-naturally occurring mutation. In another aspect, the non-naturally occurring mutation results in an increased level of activity by a protein or polypeptide encoded by a polynucleotide comprising the non-naturally occurring mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-naturally occurring mutation.

[0093] In one aspect, mutation of a genomic locus results in a reduced expression level compared to a genomic locus that lacks the mutation.In another aspect, mutation of a genomic locus results in an increased expression level compared to a genomic locus that lacks the mutation.In a further aspect, mutation of a genomic locus results in a reduced activity level by the protein or polypeptide encoded by the genomic locus with the mutation compared to the protein or polypeptide encoded by the genomic locus that lacks the mutation.In a further aspect, mutation of a genomic locus results in an increased activity level by the protein or polypeptide encoded by the genomic locus with the mutation compared to the protein or polypeptide encoded by the genomic locus that lacks the mutation.

[0094] The level of gene expression is routinely examined in the art.As a non-limiting example, gene expression can be measured using quantitative reverse transcriptase PCR (qRT-PCR), RNA sequencing or Northern blot.In one aspect, gene expression is measured using qRT-PCR.In another aspect, gene expression is measured using Northern blot.In another aspect, gene expression is measured using RNA sequencing.

[0095] In one aspect, the methods provided herein further include crossing the tobacco plant with a tobacco plant containing a mutation or transgene that directly suppresses or eliminates 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 a MATE transporter.

[0096] As used herein, "transgene" refers to foreign DNA that is stably integrated into the genome of a modified tobacco plant.

[0097] In one aspect, a tobacco plant comprising the PY trait further comprises a mutation or transgene that directly suppresses or eliminates the expression or activity of one or more genes encoding a product selected from the group consisting of methylputrescine oxidase (MPO), quinolate phosphoribosyltransferase (QPT), quinolinate synthase (QS), BBL, A622, aspartate oxidase, agmatine deiminase (AIC), S-adenosyl-methionine synthetase (SAMS), arginase, diamine oxidase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), arginine decarboxylase (ADC), nicotine uptake permease (NUP), and a MATE transporter.

[0098] In one aspect, a tobacco plant comprising a PY trait further comprises a mutation in an ERF gene at the Nic2 locus. In one aspect, a tobacco plant comprising a PY trait further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or all seven genes selected from the group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. See Shoji et al., Plant Cell, (10):3390-409 (2010); and Kajikawa et al., Plant physiol. 2017, 174:999-1011. In one aspect, a tobacco plant comprising a PY trait further comprises one or more mutations in ERF189, ERF115, or both.

[0099] In one aspect, a tobacco plant comprising a PY trait further comprises a mutation in an ERF gene at the Nic1 locus (or the Nic1b locus as in PCT / US2019 / 013345, filed January 11, 2019, published as WO / 2019 / 140297). See also WO / 2018 / 237107. In one aspect, a tobacco comprising a PY trait plant further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See Kajikawa et al., Plant physiol. 2017, 174:999-1011. In one aspect, the tobacco plant comprising the PY trait 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.

[0100] In one aspect, the tobacco plants provided herein contain a mutation or transgene that confers reduced nicotine levels compared to a tobacco plant lacking the mutation or transgene. In one aspect, the tobacco plants provided herein are low-alkaloid tobacco plants.

[0101] Various factors, including genotype, environment, fertilization, and agronomic practices, affect tobacco alkaloid levels (e.g., nicotine production is stimulated by pinching, wounding, and herbivore damage). The low-alkaloid trait, first discovered in a line of Cuban cigar tobacco, was introduced into tobacco cultivars through a series of backcrosses. Low-alkaloid tobacco germplasm was then registered in the genetic background of cultivar Burley 21 (Legg et al., Crop Science, 10:212 (1970)). Genetic studies using the low-alkaloid Burley 21 (LA BU21) line demonstrated that two unlinked loci contribute to nicotine levels in tobacco leaves. These two loci are referred to as Nic1 and Nic2. The nic1 and nic2 (same as nicotine1 and nicotine2, respectively) mutations in LA BU21 are semidominant. They show a dose-dependent effect on nicotine levels, with the effect of nic1 being approximately 2.4-fold stronger than that of nic2. Molecular characterization of the Nic2 locus has been reported. The nic2 mutation was shown to contain a deletion of a cluster of transcription factor genes from the ethylene response factor (ERF) family, e.g., ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168 (Shoji et al., Plant Cell, (10):3390-409 (2010)).

[0102] Reducing the total alkaloid content of tobacco could have many benefits. It could increase the value of tobacco as a biomass resource. Increasing nicotinic alkaloids in tobacco plants could play an important role in protecting the plant from insects and herbivores.

[0103] Consistent with the role of alkaloids in insect defense, LA BU21 was reported to be extremely susceptible to insect damage (Legg et al., Crop Science, 10:212 (1970)). Further studies comparing isogenic lines of flue-cured tobacco with low total alkaloid percentages (approximately 0.20%) to their "normal" recurrent parents (total alkaloids 1.85-2.70%) reported that yield, grade index, total N, and reducing sugar content in the low-alkaloid lines were lower than in normal flue-cured cultivars (Chaplin and Weeks, Crop Science, 16(3):416-18 (1976)).

[0104] Low alkaloid tobacco varieties include, but are not limited to, LA Burley 21, LAFC53, LN B&W, and LN KY171.

[0105] In one aspect, the mutation that confers reduced nicotine levels comprises a nic1 mutation, a nic2 mutation, or both. In one aspect, the nic1 mutation, the nic2 mutation, or both are introgressed from or derived from a variety selected from the group consisting of LA Burley 21, LAFC53, LN B&W, and LN KY171.

[0106] As used herein, a "low alkaloid variety" of tobacco refers to a tobacco variety that includes one or more genetic modifications that reduce total alkaloids (measured by dry weight) to a level that is less than 25% of the total alkaloid level in a control tobacco variety of a substantially similar genetic background except for the one or more genetic modifications. As a non-limiting example, KY171 can serve as a control for the low alkaloid variety LA KY171.

[0107] As used herein, "genetically modified" refers to plants, seeds, plant parts, plant cells, and plant genomes that have been subjected to mutagenesis, genome editing, genetic transformation, or a combination thereof.

[0108] In one aspect, the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factors, nicotine uptake permease (NUP), and a MATE transporter. In another aspect, the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus that encodes a protein selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. In another aspect, the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus that encodes a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0109] In one aspect, transgenes that confer reduced nicotine levels include transgenes that target and repress genes encoding proteins selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factors, nicotine uptake permease (NUP), and MATE transporters. In another aspect, the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. In another aspect, the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

[0110] In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49.In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49. In another aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 22-27, 44, 45, and 49.

[0111] In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the tobacco plant, seed, or cell comprises a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.

[0112] In one aspect, the tobacco plant, seed, or cell comprises a heterologous promoter operably linked to a polynucleotide comprising a non-naturally occurring mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0113] In one aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in the endogenous nucleic acid sequence encoding the g58899 protein, wherein the non-naturally occurring mutation comprises an insertion, deletion, or substitution of an amino acid residue at a position selected from the group consisting of 18, 24, 54, 56, 57, 60, 87, 221, and 325 relative to SEQ ID NO: 46.

[0114] In one aspect, a tobacco plant, seed, or cell comprises a non-naturally occurring mutation in the promoter of an endogenous nucleic acid sequence encoding a g58899 protein, wherein the tobacco plant, seed, or cell exhibits reduced expression of the endogenous nucleic acid compared to a control tobacco plant, seed, or cell lacking the non-naturally occurring mutation.

[0115] In one aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a g58899 protein, wherein the non-naturally occurring mutation results in a premature stop codon in the g58899 protein compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 46, and 47. In another aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a g58887 protein, wherein the non-naturally occurring mutation results in a premature stop codon in the g58887 protein compared to SEQ ID NO: 28. In another aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a g58888 protein, wherein the non-naturally occurring mutation results in a premature stop codon in the g58888 protein compared to SEQ ID NO: 29. In another aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a g58917 protein, wherein the non-naturally occurring mutation results in a premature stop codon in the g58917 protein compared to SEQ ID NO: 31. In another aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a g58905 protein, wherein the non-naturally occurring mutation results in a premature stop codon in the g58905 protein compared to SEQ ID NO: 32. In another aspect, the tobacco plant, seed, or cell comprises a non-naturally occurring mutation in an endogenous nucleic acid sequence encoding a g61524 protein, wherein the non-naturally occurring mutation results in a premature stop codon in the g61524 protein compared to SEQ ID NO: 33.

[0116] artificial miRNA In one aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 96% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 97% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA encoding an amino acid sequence having 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.

[0117] In one aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 85% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 96% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 97% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the disclosure provides a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule capable of binding to RNA that is 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0118] In one aspect, the non-coding RNA molecule is selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), trans-acting siRNA (ta-siRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), intron, hairpin RNA (hpRNA), and intron-containing hairpin RNA (ihpRNA).

[0119] miRNAs are typically about 19 to about 25 nucleotides long (typically about 20 to 24 nucleotides in plants) and guide the trans cleavage of target transcripts, negatively regulating the expression of genes involved in various regulatory and developmental pathways (Bartel (2004) Cell, 116:281-297). In some cases, miRNAs serve to guide the in-phase processing of primary transcripts into siRNAs (see Allen et al. (2005) Cell, 121:207-221).

[0120] Many microRNA genes (MIR genes) have been identified and published in databases ("miRBase," available online at microrna[dot]sanger[dot]ac[dot]uk / sequences; see also Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). MIR genes have been reported to occur in isolated and clustered intergenic regions of the genome, but they can also be located wholly or partially within introns of other genes (both protein-coding and non-protein-coding). For a recent review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Transcription of MIR genes can, at least in some cases, be under the regulating control of the MIR gene's own promoter. The primary transcript, termed the "pri-miRNA," is fairly large (several kilobases), contains one or more pre-miRNAs (a foldback structure containing a stem-loop sequence that is processed into a mature miRNA), as well as the usual 5' "cap" and polyadenylated tail of an mRNA, and may be polycistronic. See, e.g., Figure 1 in Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385.

[0121] The maturation of mature miRNAs from their corresponding precursors (pri-miRNA and pre-miRNA) differs significantly between animals and plants. For example, in plant cells, microRNA precursor molecules are thought to be primarily processed into mature miRNAs exclusively in the nucleus, whereas in animal cells, pri-miRNA transcripts are processed in the nucleus by the animal-specific enzyme Drosha, followed by transport of the pre-miRNA to the cytoplasm, where it is further processed into mature miRNAs. Mature miRNAs in plants are typically 21 nucleotides in length. For a recent review of miRNA biogenesis in both plants and animals, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Further reviews of miRNA biogenesis and function can be found, for example, in Bartel (2004) Cell, 116:281-297; Murchison and Hannon (2004) Curr. Opin. Cell Biol., 16:223-229; and Dugas and Bartel (2004) Curr. Opin. Plant Biol., 7:512-520.

[0122] Transgenic expression of miRNA (whether naturally occurring sequence or artificial sequence) can be used to regulate the expression of the target gene of miRNA.The miRNA recognition site contained in transgenically expressed transcript is also useful for regulating the expression of transcript; for example, see Parizotto et al. (2004) Genes Dev., 18:2237-2242. miRNA recognition sites have been validated in all regions of mRNAs, including the 5' untranslated, coding, and 3' untranslated regions, suggesting that the location of miRNA target sites relative to the coding sequence may not necessarily affect repression (see, e.g., Jones-Rhoades and Bartel (2004). Mol. Cell, 14:787-799; Rhoades et al. (2002) Cell, 110:513-520; Allen et al. (2004) Nat. Genet., 36:1282-1290; Sunkar and Zhu (2004) Plant Cell, 16:2001-2019). Because miRNAs are important regulatory elements in eukaryotes, transgenic silencing of miRNAs is useful for manipulating biological pathways and responses. Finally, promoters of MIR genes can have highly specific expression patterns (e.g., cell-specific, tissue-specific, time-specific, or inducible) and are therefore useful in recombinant constructs to direct such specific transcription of DNA sequences to which they are operably linked. The various utilities of miRNAs, their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication No. 2006 / 0200878 A1, which is incorporated herein by reference.Non-limiting examples of these utilities include: (1) expression of native miRNA or miRNA precursor sequences to repress target genes; (2) expression of artificial miRNA or miRNA precursor sequences to repress target genes; (3) expression of transgenes containing miRNA recognition sites, where expression of the mature miRNA results in repression of the transgene; and (4) expression of transgenes driven by miRNA promoters.

[0123] Designing an artificial miRNA sequence can be as simple as replacing nucleotides in the miRNA stem region of a miRNA precursor with a sequence complementary to the intended target, as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333. One non-limiting example of a general method for determining nucleotide changes in a native miRNA sequence to produce an engineered miRNA precursor includes the following steps: (a) selecting a unique target sequence of at least 18 nucleotides specific to the target gene, for example, from both tobacco cDNA and genomic DNA databases, by using a sequence alignment tool such as BLAST (see, e.g., Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402), to identify all potential matches with the target transcript ortholog and unrelated genes, thereby avoiding unintended silencing of non-target sequences; (b) analyzing the target gene for undesired sequences (e.g., matches with sequences from non-target species), and determining the sequence of the target gene based on GC content, Reynolds score (Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and functional asymmetry characterized by a negative difference in free energy (".DELTA..DELTA.G" or "ΔΔG") (see Khvorova et al. (2003) Cell, 115:209-216). Preferably, 19-mers are selected that have all or most of the following characteristics: (1) a Reynolds score >4, (2) a GC content of about 40% to about 60%, (3) a negative ΔΔG, (4) a terminal adenosine, (5) lack of consecutive runs of four or more identical nucleotides, (6) a location near the 3' end of the target gene; and (7) minimal differences from the pre-mRNA transcript.The position of every third nucleotide in an siRNA has been reported to be particularly important in influencing RNAi efficacy, and the algorithm "siExplorer" is published at rna[dot]chem[dot]t[dot]u-tokyo[dot]ac[dot]jp / siexplorer.htm (see Katoh and Suzuki (2007) Nucleic Acids Res., 10.1093 / nar / gkl1120); (c) determining the reverse complement of the selected 19-mer for use in generating a modified mature miRNA. The additional nucleotide at position 20 preferably matches the selected target sequence, and the nucleotide at position 21 is preferably selected to be either unpaired to prevent spread of silencing on the target transcript or paired with the target sequence to promote spread of silencing on the target transcript; and (d) transforming the artificial miRNA into a plant.

[0124] In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 80% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 85% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 95% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 96% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 97% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 98% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNAs provided herein are complementary to a polynucleotide having at least 99% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48. In one aspect, the artificial miRNA provided herein is complementary to a polynucleotide having 100% sequence identity with a polynucleotide selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0125] In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 80% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 85% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 90% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 95% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 96% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 97% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 98% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having at least 99% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In another aspect, the artificial miRNAs provided herein are complementary to a polynucleotide encoding a polypeptide having 100% sequence identity or similarity to a polypeptide selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.

[0126] In one aspect, an artificial miRNA comprises at least 15 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, an artificial miRNA comprises at least 16 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, an artificial miRNA comprises at least 17 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, an artificial miRNA comprises at least 18 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, an artificial miRNA comprises at least 19 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the artificial miRNA comprises at least 20 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. In another aspect, the artificial miRNA comprises at least 21 contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

[0127] In one aspect, the artificial miRNAs provided herein reduce or eliminate RNA transcription or protein translation of a gene selected from the group consisting of G58887, g58888, g58899, g58917, g58905, g61524, and g20337.

[0128] In one aspect, the modified tobacco plant or part thereof comprises any of the non-coding RNA molecules provided herein. In another aspect, the modified tobacco plant or part thereof comprises any of the artificial miRNAs provided herein. In another aspect, the modified tobacco plant or part thereof comprises any of the recombinant nucleic acids provided herein.

[0129] When expressed in tobacco plants, the non-coding RNA molecules provided herein reduce the expression or translation of their cognate target transcripts (e.g., SEQ ID NOs: 16-21 and 48) compared to control tobacco plants that do not express the non-coding RNA molecule. Similarly, when expressed in tobacco plants, the artificial miRNAs provided herein reduce the expression or translation of their cognate target transcripts (e.g., SEQ ID NOs: 16-21 and 48) compared to control tobacco plants that do not express the non-coding RNA molecule.

[0130] In one aspect, the modified tobacco plants provided herein exhibit a buff phenotype.

[0131] In one aspect, the artificial miRNA is operably linked to a heterologous promoter.

[0132] As used herein, "capable of binding" is synonymous with "capable of hybridizing with." In one aspect, a first nucleic acid molecule capable of binding to a second nucleic acid molecule binds to the second nucleic acid molecule. As used herein, a first nucleic acid molecule can "hybridize" a second nucleic acid molecule in a sequence-specific, antiparallel manner through non-covalent interactions (e.g., Watson-Crick base pairing) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength (i.e., a nucleic acid specifically binds to a complementary nucleic acid). As is known in the art, standard Watson-Crick base pairing includes adenine pairing with thymine, adenine pairing with uracil, and guanine (G) pairing with cytosine (C) [DNA, RNA]. Furthermore, in the case of hybridization between two RNA molecules (e.g., dsRNA), it is also known in the art that guanine bases pair with uracil. For example, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pairing with codons in mRNA.In the context of the present disclosure, the guanine of the protein binding segment (dsRNA duplex) of the subject DNA-targeting RNA molecule is considered to be complementary to uracil, and vice versa.Therefore, if a G / U base pair can be created at a given nucleotide position of the protein binding segment (dsRNA duplex) of the subject DNA-targeting RNA molecule, this position is not considered to be non-complementary, but instead is considered to be complementary.

[0133] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the "stringency" of hybridization.

[0134] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. Suitable conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables well known in the art. The higher the degree of complementarity between two nucleotide sequences, the higher the melting temperature (Tm) of hybrids of nucleic acids having those sequences. For hybridization between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or fewer nucleotides), the location of mismatches becomes important (see Sambrook et al.). Typically, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Exemplary minimum lengths for a hybridizable nucleic acid are at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides. Furthermore, those skilled in the art will recognize that the temperature and salt concentration of the wash solution can be adjusted as needed depending on factors such as the length of the complementary region and the degree of complementarity.

[0135] It is understood in the art that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable.Furthermore, a polynucleotide can hybridize across one or more segments, such that intervening or adjacent segments are not involved in hybridization (for example, a loop structure or a hairpin structure).For example, an antisense nucleic acid in which 18 of the 20 nucleotides of an antisense compound are complementary to a target region and therefore will specifically hybridize will represent 90% complementarity.In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides, and do not need to be adjacent to each other or complementary nucleotides. The percent complementarity between particular stretches of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST® program (Basic Local Alignment Search Tool) and PowerBLAST program known in the art (see Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings that employ the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489).

[0136] promoter As generally understood in the art, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box and that supports or facilitates the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters that contain a combination of two or more heterologous sequences. Thus, promoters of the present application can include variants of promoter sequences that are similar in composition, but not identical, to those provided herein or to other known promoter sequences.

[0137] A promoter that drives expression in all or most tissues of a plant is referred to as a "constitutive" promoter. A non-limiting example of a constitutive promoter is the Cauliflower Mosaic Virus (CaMV) 35S promoter. A promoter that drives expression during a specific period or stage of development is referred to as a "developmental" promoter. A promoter that drives enhanced expression in specific tissues of an organism relative to other tissues of the organism is referred to as a "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively high or preferential expression in specific tissues of a plant, but causes lower levels of expression in other tissues of the plant. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli such as heat, cold, drought, light, or other stimuli such as wounding or application of chemicals.

[0138] In one aspect, the promoters provided herein are constitutive promoters. In another aspect, the promoters provided herein are inducible promoters. In a further aspect, the promoters provided herein are developmental promoters.

[0139] In one aspect, the present disclosure provides a heterologous promoter. In another aspect, the present disclosure provides a promoter operably linked to a heterologous polynucleotide. In another aspect, the present disclosure provides a polynucleotide sequence operably linked to a heterologous promoter.

[0140] As used herein, "operably linked" refers to the functional link between two or more elements.For example, the functional link between a polynucleotide of interest and a regulatory sequence (e.g., promoter) is the functional link that allows the expression of the polynucleotide of interest.The functionally linked elements may be adjacent or not adjacent.In one aspect, the promoter provided herein is functionally linked to a heterologous nucleic acid molecule.

[0141] plant As used herein, "tobacco" refers to Nicotiana tabacum.

[0142] In one aspect, the tobacco parts provided include, but are not limited to, leaves, stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, fruits, meristems, cotyledons, hypocotyls, pods, embryos, endosperm, explants, callus, tissue cultures, shoots, cells, and protoplasts. In one aspect, the tobacco parts provided do not include seeds. In one aspect, the present disclosure provides tobacco plant cells, tissues, and organs that are not reproductive material and do not mediate natural plant reproduction. In another aspect, the present disclosure also provides tobacco plant cells, tissues, and organs that are reproductive material and mediate natural plant reproduction. In another aspect, the present disclosure provides tobacco plant cells, tissues, and organs that cannot sustain themselves through photosynthesis. In another aspect, the present disclosure provides somatic tobacco plant cells. Somatic cells, in contrast to reproductive cells, do not mediate plant reproduction.

[0143] The cells, tissues, and organs can be derived from seeds, fruits, leaves, cotyledons, hypocotyls, meristems, embryos, endosperms, roots, shoots, stems, pods, flowers, inflorescences, stems, pedicels, styles, stigmas, receptacles, petals, sepals, pollen, anthers, filaments, ovaries, ovules, pericarp, phloem, and vascular tissues. In another aspect, the present disclosure provides tobacco plant chloroplasts. In a further aspect, the present disclosure provides epidermal cells, stomatal cells, leaves or root hairs, storage roots, or tubers. In another aspect, the present disclosure provides tobacco protoplasts.

[0144] Those skilled in the art understand that tobacco plants naturally propagate through seeds, rather than through asexual or vegetative propagation. In one aspect, the present disclosure provides tobacco endosperm.

[0145] The present disclosure provides cells from the tobacco plants provided herein.

[0146] As used herein, a "progeny plant" can be derived from any hybrid generation, e.g., F1, F2, F3, F4, F5, F6, F7, etc.

[0147] In one aspect, the tobacco plant is of a tobacco variety selected from the group consisting of flue-cured, bright, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish. In one aspect, the modified tobacco plant provided herein is of a tobacco variety selected from the group consisting of flue-cured, bright, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish.

[0148] In one aspect, the tobacco cells are of a tobacco variety selected from the group consisting of Flucured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish. In one aspect, the modified tobacco cells are of a tobacco variety selected from the group consisting of Flucured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish.

[0149] In one aspect, the tobacco leaf is of a tobacco variety selected from the group consisting of Flue-Cured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish.

[0150] In one aspect, the cured tobacco leaves or plant parts are of a tobacco variety selected from the group consisting of Flue-Cured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish. Those skilled in the art will further understand that cured tobacco does not constitute a living organism and is incapable of growth or reproduction.

[0151] Flue-cured tobacco (also known as "Virginia" or "bright" tobacco) accounts for approximately 40% of the world's tobacco production. Flue-cured tobacco is often referred to as "bright tobacco" because it turns golden to deep orange during curing. Flue-cured tobacco has a light, bright aroma and flavor. Flue-cured tobacco is generally high in sugar and low in oil. Major growing countries for flue-cured tobacco are Argentina, Brazil, China, India, Tanzania, and the United States. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2 and any variety essentially derived from any one of the foregoing varieties. See WO 2004 / 041006 A1. In a further aspect, the modified tobacco plant or seed provided herein is of a flue-cured variety selected from the group consisting of K326, K346, and NC196.

[0152] (Table 2) Flue-cured tobacco varieties TIFF2026035667000029.tif166150TIFF2026035667000030.tif162150

[0153] Air-cured tobacco includes "burley," "Maryland," and "dark" tobacco. The common factor that unites air-cured tobaccos is that the curing is primarily done without heat and humidity from artificial sources. Burley tobacco is light to dark brown in color and is high in oil and low in sugar. Burley tobacco is typically air-cured in warehouses. Major burley-growing countries include Argentina, Brazil, Italy, Malawi, and the United States.

[0154] Maryland tobacco is extremely fluffy, has good burning characteristics, low nicotine and a neutral flavor. Major growing countries for Maryland include the United States and Italy.

[0155] In one aspect, the tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a Burley tobacco variety selected from the group consisting of the tobacco varieties set forth in Table 3, and any variety essentially derived from any one of the foregoing varieties. In a further aspect, the modified tobacco plants or seeds provided herein are of a Burley variety selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488.

[0156] (Table 3) Burley tobacco varieties TIFF2026035667000031.tif197166

[0157] In another aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a Maryland tobacco variety selected from the group consisting of the tobacco varieties set forth in Table 4, and any variety essentially derived from any one of the foregoing varieties.

[0158] Table 4. Maryland Tobacco Varieties TIFF2026035667000032.tif73128

[0159] Dark air-cured tobacco is distinguished from other tobacco types primarily by its curing process, which gives dark air-cured tobacco its medium to dark brown color and distinctive aroma. Dark air-cured tobacco is primarily used in the production of chewing tobacco and snuff. In one aspect, the modified tobacco plants or seeds provided herein are of a dark air-cured tobacco variety selected from the group consisting of Sumatra, Jatim, Dominican Cubano, Besuki, One Sucker, Green River, Virginia Sun-cured, and Paraguan Passado, and any variety essentially derived from any one of the foregoing varieties.

[0160] Dark fire-cured tobacco is typically dried over a low-burning wood fire on the floor of a closed dry barn. Dark fire-cured tobacco is typically used to make pipe blends, cigarettes, chewing tobacco, snuff, and strong-tasting cigars. The primary growing regions for dark fire-cured tobacco are Tennessee, Kentucky, and Virginia in the United States. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a dark fire-cured tobacco variety selected from the group consisting of the tobacco varieties listed in Table 5 and any variety essentially derived from any one of the foregoing varieties.

[0161] Table 5. Dark Fire-Cured Tobacco Varieties TIFF2026035667000033.tif183151

[0162] Oriental tobacco is also referred to as Greek tobacco, flavored tobacco, and Turkish tobacco due to the fact that it is commonly grown in eastern Mediterranean regions such as Turkey, Greece, Bulgaria, Macedonia, Syria, Lebanon, Italy, and Romania. The small plant size, small leaf size, and unique flavor characteristics of Oriental tobacco varieties are the result of their adaptation to the poor soils and stressful climatic conditions in which they were developed. In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of an Oriental tobacco variety selected from the group consisting of the tobacco varieties listed in Table 6 and any variety essentially derived from any one of the aforementioned varieties.

[0163] Table 6. Oriental Tobacco Varieties TIFF2026035667000034.tif180144

[0164] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a cigar tobacco variety selected from the group consisting of the tobacco varieties set forth in Table 7, and any variety essentially derived from any one of the foregoing varieties.

[0165] (Table 7) Cigar Tobacco Varieties TIFF2026035667000035.tif74162

[0166] In one aspect, the tobacco plant or seed or modified tobacco plant or seed provided herein is of a tobacco variety selected from the group consisting of the tobacco varieties set forth in Table 8, and any variety essentially derived from any one of the foregoing varieties.

[0167] (Table 8) Other tobacco varieties TIFF2026035667000036.tif36128

[0168] In one aspect, the tobacco plant, seed, or cell is from a variety selected from the group consisting of the tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.

[0169] Any particular varieties of the aforementioned fluid-cured, dark air-cured, burley, Maryland, dark fire-cured, cigar, or Oriental types are listed for illustrative purposes only, and any additional fluid-cured, dark air-cured, burley, Maryland, dark fire-cured, cigar, or Oriental varieties are also contemplated herein.

[0170] In one aspect, the tobacco plants or varieties provided herein are inbred tobacco plants or varieties. As used herein, an "inbred" tobacco variety is a variety that has been bred for genetic homogeneity.

[0171] In one aspect, the tobacco plants or varieties provided herein are hybrid tobacco plants or varieties. As used herein, a "hybrid" is produced by crossing two plants from different varieties or species so that the offspring contain genetic material from each parent. Those skilled in the art will recognize that higher-order hybrids can also be produced. For example, a first hybrid can be produced by crossing variety C with variety D to produce a C x D hybrid, and a second hybrid can be produced by crossing variety E with variety F to produce an E x F hybrid. The first and second hybrids can be further crossed to produce a higher-order hybrid (C x D) x (E x F) that contains genetic information from all four parent varieties. In one aspect, the modified tobacco plants provided herein are hybrid tobacco plants. In another aspect, the modified tobacco seeds provided herein are hybrid tobacco seeds.

[0172] As used herein, the term "crossing" refers to the intentional mating of two plants. In one aspect, crossing includes pollination and / or fertilization of a first tobacco plant with a second tobacco plant. The two tobacco plants being crossed can be distantly related, closely related, or identical. In one aspect, both of the two tobacco plants being crossed are modified tobacco plants. In one aspect, the two tobacco plants being crossed are of the same tobacco variety. In one aspect, the two tobacco plants being crossed are of two different tobacco varieties. In one aspect, one of the two tobacco plants being crossed is male sterile. In one aspect, one of the two tobacco plants being crossed is female sterile. In one aspect, at least one of the two tobacco plants being crossed is a hybrid tobacco plant. In one aspect, at least one of the two tobacco plants to be crossed is a modified tobacco plant.

[0173] In one aspect, the tobacco plants or varieties provided herein are male sterile. In another aspect, the tobacco plants or varieties provided herein are cytoplasmic male sterile (CMS). Male sterile tobacco plants can be produced by any method known in the art. The method for producing male sterile tobacco is described in Wernsman, EA, and Rufty, RC 1987. Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. Crop Species. WH Fehr (ed.), MacMillan Publishing Go., Inc., New York, NY 761 pp.

[0174] In another aspect, the tobacco plant or variety provided herein is female sterile.By way of non-limiting example, female sterile plants can be produced by mutating STIG1 gene.See, for example, Goldman et al.1994, EMBO Journal 13:2976-2984.In one aspect, the modified tobacco plant provided herein is female sterile.

[0175] As used herein, a "population" of a plant or seed refers to a set of any number of individuals, objects, or data from which samples are collected for evaluation, including one. Most commonly, this term refers to a breeding population of plants whose members are selected and crossed to produce progeny in a breeding program. A plant population can include the progeny of a single breeding cross or multiple breeding crosses, and can be either actual plants or plant-derived materials, or in silico representations of plants or seeds. Population members do not need to be identical to the population members selected for use in subsequent analysis cycles, or the population members ultimately selected to obtain the final progeny plants or seeds. Often, a plant or seed population is derived from a single biparental cross, but it can also be derived from two or more crosses between the same or different parents. A plant or seed population can contain any number of individuals, but those skilled in the art will recognize that plant breeders typically use population sizes ranging from 100 or 200 individuals to several thousand individuals, and that the highest performing 5% to 20% of the population are typically selected for use in subsequent crosses to improve the performance of subsequent generations of the population.

[0176] Numerous methods for introducing recombinant DNA constructs into plant cells are known in the art and can be used by the methods of the present application to produce transgenic plant cells and plants. Any suitable method or technique for transforming plant cells known in the art can be used by the present methods. Effective methods for plant transformation include bacterial-mediated transformation, such as Agrobacterium- or Rhizobium-mediated transformation, and microprojectile bombardment-mediated transformation. Various methods are known in the art for regenerating or developing transgenic plants, such as by transforming explants with transformation vectors via bacterial-mediated transformation or microprojectile bombardment, followed by culturing the explants. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, polyethylene glycol (PEG)-mediated transformation, and the like, are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric with respect to the transformation event, depending on the method and explant used.

[0177] The method of transforming plant cells is well known to those skilled in the art.For example, the specific instructions for transforming plant cells by microprojectile bombardment with particles coated with recombinant DNA (for example, biolistic transformation) can be found in United States Patent No. 5,550,318; United States Patent No. 5,538,880; United States Patent No. 6,160,208; United States Patent No. 6,399,861; and United States Patent No. 6,153,812, and Agrobacterium-mediated transformation is described in United States Patent No. 5,159,135; United States Patent No. 5,824,877; United States Patent No. 5,591,616; United States Patent No. 6,384,301; United States Patent No. 5,750,871; United States Patent No. 5,463,174; and United States Patent No. 5,188,958, all of which are incorporated herein by reference. Further methods for transforming plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those of skill in the art can be used to transform tobacco cells with any of the nucleic acid molecules provided herein.

[0178] In one aspect, the method for providing a nucleic acid molecule to a tobacco cell comprises Agrobacterium-mediated transformation. In another aspect, the method for providing a nucleic acid molecule to a cell comprises PEG-mediated transformation. In another aspect, the method for providing a nucleic acid molecule to a cell comprises biolistic transformation. In another aspect, the method for providing a nucleic acid molecule to a cell comprises liposome-mediated transfection (lipofection). In another aspect, the method for providing a nucleic acid molecule to a cell comprises lentiviral transfection.

[0179] Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in WO 91 / 17424 and WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0180] Any tobacco cell that can regenerate fertile tobacco plants is contemplated as a recipient cell useful for practicing the present disclosure. In one aspect, a recombinant DNA construct is introduced into a tobacco cell. In one aspect, a recombinant DNA construct is introduced into a tobacco protoplast cell. In another aspect, a recombinant DNA construct is introduced into a tobacco callus cell. In one aspect, a recombinant DNA construct is introduced into a tobacco cell selected from the group consisting of seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, flower cells, inflorescence cells, stem cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, and phloem cells.

[0181] Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or embryo parts, seedling apical meristems, microspores, etc. Those cells that can grow as callus can serve as recipient cells for transformation. Practical transformation methods and materials for producing the transgenic plants of the present disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication No. 2004 / 0216189, all of which are incorporated herein by reference.

[0182] TSNA reduction "Alkaloids" are complex nitrogen-containing compounds naturally occurring in plants that have pharmacological effects in humans and animals. "Nicotine" is the major natural alkaloid in commercial cigarettes, accounting for approximately 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-methylanatabine, N-methylanabasine, pseudooxynicotine, and 2,3-dipyridyl.

[0183] Alkaloid levels can be assayed by methods known in the art, such as gas-liquid chromatography, high-performance liquid chromatography, radioimmunoassay, and enzyme-linked immunosorbent assay-based quantification. For example, nicotinic alkaloid levels can be measured by a GC-FID method based on CORESTA Recommended Method No. 7, 1987 and ISO standard (ISO TC 126N 394 E). For a gas-liquid chromatography method using a capillary column and an FID detector, see also Hibi et al., Plant Physiology 100: 826-35 (1992).

[0184] Alternatively, tobacco total alkaloids can be measured using a segmented-flow colorimetric method developed for the analysis of tobacco samples, adapted by Skalar Instrument Co. (West Chester, PA) and described by Collins et al., Tobacco Science 13:79-81 (1969). Briefly, tobacco samples are dried, crushed, and extracted prior to analysis for total alkaloids and reducing sugars. The method then utilizes acetic acid / methanol / water extraction and charcoal for decolorization. Total alkaloid determination was based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of aromatic amines to form a colored complex that was measured at 460 nm.

[0185] In one aspect, levels of total or individual TSNAs are measured on lyophilized dry leaf samples using liquid chromatography with tandem mass spectrometry (LC / MS / MS).

[0186] In another aspect, introgression of the PY QTL or PY trait into a second tobacco variety reduces the level of at least one tobacco-specific nitrosamine (TSNA) compared to a second tobacco variety lacking the PY QTL or PY trait, including N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB).

[0187] In one aspect, the reduced level of at least one TSNA comprises reduced NNN. In one aspect, the reduced level of at least one TSNA comprises reduced NNK. In one aspect, the reduced level of at least one TSNA comprises reduced NAT. In one aspect, the reduced level of at least one TSNA comprises reduced NAB.

[0188] In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 1% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 2% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 3% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 4% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 5% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 15% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 25% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by at least 75% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 99% compared to a control tobacco variety lacking the PY QTL or PY trait.In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 90% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 80% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 70% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 60% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 40% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 30% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNN by 1% to 5% compared to a control tobacco variety lacking a PY QTL or PY trait.

[0189] In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 1% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 2% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 3% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 4% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 5% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 15% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 25% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by at least 75% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 99% compared to a control tobacco variety lacking the PY QTL or PY trait.In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 90% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 80% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 70% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 60% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 40% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 30% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NNK by 1% to 5% compared to a control tobacco variety lacking a PY QTL or PY trait.

[0190] In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 1% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 2% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 3% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 4% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 5% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 15% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 25% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by at least 75% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 99% compared to a control tobacco variety lacking the PY QTL or PY trait.In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 90% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 80% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 70% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 60% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 40% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 30% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAT by 1% to 5% compared to a control tobacco variety lacking a PY QTL or PY trait.

[0191] In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 1% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 2% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 3% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 4% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 5% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 15% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 25% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by at least 75% compared to a control tobacco variety lacking the PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 99% compared to a control tobacco variety lacking the PY QTL or PY trait.In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 90% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 80% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 70% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 60% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 50% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 40% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 30% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 20% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 10% compared to a control tobacco variety lacking a PY QTL or PY trait. In one aspect, the reduced level of at least one TSNA comprises a reduction in NAB by 1% to 5% compared to a control tobacco variety lacking a PY QTL or PY trait.

[0192] Leaf Grading In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index of the progeny plants compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.

[0193] In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 1% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 2% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 3% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 4% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 5% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 10% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 15% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 20% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 25% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 30% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 50% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 75% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 100% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 200% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 20% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 30% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 40% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 50% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 60% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 70% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 80% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 90% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 100% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 150% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 200% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1% to 300% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 10% to 50% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 10% to 30% compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.

[0194] In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 1 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 2 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 3 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 4 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 5 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 6 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 7 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 8 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 9 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 10 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 11 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 12 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 13 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 14 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 15 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 16 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 17 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 18 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 19 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 20 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 25 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by at least 30 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 40 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by at least 50 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1 to 90 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1 to 80 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1-70 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by 1 to 60 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by 1 to 50 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low alkaloid tobacco variety improves the USDA leaf grade index by 1 to 40 compared to a low alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1 to 30 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1 to 20 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1 to 10 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 1 to 5 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 5 to 10 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 5 to 15 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait. In one aspect, introgression of a PY QTL or PY trait into a low-alkaloid tobacco variety improves the USDA leaf grade index by 10-20 compared to a low-alkaloid tobacco variety lacking the PY QTL or PY trait.

[0195] As used herein, "low alkaloid variety" refers to a tobacco variety that contains alkaloid levels equivalent to, less than, or no more than 20% higher than those measured in known low alkaloid lines such as CS15 and LN KY171.

[0196] As used herein, "USDA Grade Index," "Grade Index," or "Numerical Grade Index" refers to the subdivision of types by group, quality, and color. In one aspect, the USDA Grade Quality Score is quantified as a graded numerical representation from 0 to 100 determined by an official USDA grade judge and is a weighted average of all stem positions. A higher Grade Index indicates higher quality. Alternatively, leaf grade can be determined via hyperspectral imaging. See, e.g., WO 2011 / 027315 (published March 10, 2011, and incorporated by reference in its entirety).

[0197] As used herein, a "certified tobacco grader" refers to a person trained to grade tobacco according to the USDA official standard grades as defined by the Agricultural Marketing System published by the United States Department of Agriculture (USDA), 7 CFR § 29. As used herein, an "official USDA grade" may be assigned by an employee, former employee, or person otherwise trained to grade tobacco according to the USDA official standard grades. An exemplary procedure for the Commercial Inspection Service's standard operations begins with a grower releasing tobacco to market, which is then arranged in flat baskets as lots. Each lot is weighed and then inspected by an official USDA grader. After inspection, the grader assigns each lot a grade, which becomes a grade certificate indicating the group, quality, and color. The procedures for grading experimental lots are similar; however, the experimental tobacco is not marketed or used for commercial purposes.

[0198] Tobacco grades are assessed based on factors including, but not limited to, leaf stem position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to the intensity and depth of leaf coloration and brilliance), hygroscopicity (the tobacco leaf's ability to absorb and retain ambient moisture), and green hue or shade. Leaf grades can be determined, for example, using official standard grades issued by the U.S. Department of Agriculture's Agricultural Marketing Service (7 U.S.C. §511). For example, official standard grades for burley tobacco (U.S. Type 31, foreign Type 93), effective November 5, 1990 (55 FR 40645); official standard grades for fluid-cured tobacco (U.S. Types 11, 12, 13, and 14, and foreign Type 92), effective March 27, 1989 (54 FR 7925); official standard grades for Pennsylvania seed leaf tobacco (U.S. Type 41), effective January 8, 1965 (29 FR 16854); official standard grades for Ohio cigar leaf tobacco (U.S. Types 42, 43, and 44), effective December 8, 1963 (28 FR 11719 and 28 FR 11926); official standard grades for Wisconsin cigar binder tobacco (U.S. Types 54 and 55), effective December 8, 1963 (28 FR 11719 and 28 FR 11926). See Official Standard Grades for Wisconsin Cigar Binder Tobacco (U.S. Types 54 and 55), effective November 20, 1969 (34 FR 17061); Official Standard Grades for Georgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62), effective April 1971. USDA Grade Index values ​​may be determined according to industry-accepted grading indices.See, e.g., Bowman et al., Tobacco Science, 32:39-40 (1988); Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated by reference in their entirety).

[0199] Unless otherwise specified, the leaf grade index value, alkaloid, or nicotine level measurements referred to herein for tobacco plants, varieties, cultivars, or lines refer to average measurements, including, for example, the average of multiple leaves from a single plant, or the average measurement from a population of tobacco plants from a single variety, cultivar, or line. The population of tobacco plants or collection of tobacco leaves used to determine the average measurement (e.g., leaf grade or alkaloid or nicotine level) can be of any size, for example, 5, 10, 15, 20, 25, 30, 35, 40, 50, or more. To determine the standard deviation, a population of at least five or more tobacco plants is used. To determine the average measurement or grade index value, standard protocols accepted by the industry are followed.

[0200] As used herein, "USDA Graded Leaf Group," "Leaf Group," or "Group" is a type of classification that encompasses closely related grades based on certain characteristics related to stem position, body, or general quality. Group is the first factor in USDA grade. Group determination is part of the grading procedure and is assigned by official USDA grade judges.

[0201] Unless otherwise specified, measurements of alkaloid or nicotine levels (or another leaf chemistry or property characterization) or leaf grade index values ​​referred to herein for a tobacco plant, variety, cultivar, or line refer to average measurements, including, for example, the average of multiple leaves from a single plant, or the average measurement from a population of tobacco plants derived from a single variety, cultivar, or line.

[0202] Unless otherwise specified, nicotine or alkaloid levels (or another leaf chemistry or property characterization) are measured post-pick in pooled leaf samples collected from post-pick leaf numbers 3, 4, and 5. As used herein, whenever a comparison of leaves from two plants (e.g., mutant plant vs. control plant) is referred to, leaves from the same or equivalent stem position and developmental stage are intended so that the comparison can demonstrate effects due to genotypic differences rather than other factors. As a non-limiting example, leaf 3 of a wild-type control plant is intended as a reference point for comparison with leaf 3 of a plant containing the PY trait.

[0203] As used herein, leaf numbering is based on the leaf's position on the tobacco stem, with leaf number 1 being the youngest leaf (top) after topping and the highest leaf number being assigned to the oldest leaf (bottom).

[0204] Unless otherwise specified, all comparisons with a control plant require similar or equivalent growing conditions for the two plants being compared. As used herein, "similar growing conditions" or "equivalent growing conditions" refers to similar growing conditions and / or agricultural practices for growing and meaningfully comparing two or more plant genotypes such that the environmental conditions or agricultural practices do not contribute to or account for any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water (humidity), and nutrients (e.g., nitrogen and phosphorus). Agricultural practices include, for example, sowing, pruning, undercutting, transplanting, topping, and division. See Chapters 4B and 4C of Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp. 70-103.

[0205] drying / product "Curing" is a ripening process that reduces moisture and causes the breakdown of chlorophyll, giving tobacco leaves their golden color and thereby converting starch to sugars. Cured tobacco therefore has a higher reducing sugar content and a lower starch content compared to harvested green leaves. In one aspect, the tobacco plants or plant components provided herein can be dried using conventional means, such as 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 typically aged in a compressed state in wooden drums (e.g., vats) or cardboard boxes for several years (e.g., 2-5 years) at moisture contents ranging from 10% to about 25%. See U.S. Patent Nos. 4,516,590 and 5,372,149. The cured and aged tobacco can then be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation.

[0206] Information regarding the harvesting of burley and dark tobacco varieties can be found in the 2019-2020 Burley and Dark Tobacco Production Guide (December 2018), published by the University of Kentucky, the University of Tennessee, Virginia Polytechnic Institute and University, and North Carolina State University, which is incorporated herein by reference in its entirety.

[0207] In one aspect, the present disclosure provides a cured tobacco material from any of the plants provided herein.

[0208] In one aspect, the cured tobacco material comprises a tobacco material selected from the group selected from leaf material, stem material, bud material, flower material, and root material.

[0209] In one aspect, the cured tobacco provided herein is selected from the group consisting of air-cured tobacco, flue-cured tobacco, sun-cured tobacco, and flue-cured tobacco. In another aspect, the cured tobacco material provided herein is selected from the group consisting of air-cured tobacco, flue-cured tobacco, sun-cured tobacco, and flue-cured tobacco. In one aspect, the cured tobacco is derived from a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Oriental, and Turkish. In another aspect, the cured tobacco material is derived from a tobacco variety selected from the group consisting of flue-cured, bright, burley, Virginia, Maryland, dark, Oriental, and Turkish.

[0210] Fermentation is typically characterized by a high initial moisture content, heat generation, and a loss of 10-20% dry weight. See, e.g., U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, and 5,372,149; U.S. Patent Application Publication No. 2005 / 0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, edited by Davis & Nielsen, Blackwell Publishing, Oxford). The cured, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, e.g., U.S. Pat. Nos. 4,528,993, 4,660,577, and 4,987,907. In one aspect, the present disclosure provides a fermented tobacco material derived from any of the tobacco plants provided herein.

[0211] Tobacco materials 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 intended for human use or consumption. In one aspect, the present disclosure provides a tobacco product comprising plant material from the tobacco plant provided herein. In another aspect, the present disclosure provides a tobacco product comprising dried tobacco material. In another aspect, the present disclosure provides a tobacco product comprising fermented tobacco material.

[0212] Tobacco products provided include, but are not limited to, cigarette products (e.g., cigarettes and bidi cigarettes), cigars (e.g., cigars and cigarillos), pipe tobacco products, tobacco-derived products, tobacco-derived nicotine products, smokeless tobacco products (e.g., moist snuff, dry snuff, and chewing tobacco), films, chewables, tablets (tabs), molded parts, gels, consumable units, insoluble matrices, hollow shapes, reconstituted tobacco, expanded tobacco, etc. See, e.g., U.S. Patent Application Publication No. 2006 / 0191548.

[0213] As used herein, "cigarette" refers to a tobacco product having a "rod" and "filler." The cigarette "rod" includes the cigarette paper, filter, plug wrap (used to contain the filtration material), tipping paper that holds the cigarette paper (containing the filler) to the filter, and any adhesives that hold these components together. "Filler" includes (1) all tobacco, 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 tobacco wrapped in cigarette paper); (3) packaging materials; (4) flavorings; and (5) all other additives (mixed with tobacco and substitutes and rolled into a cigarette).

[0214] As used herein, "reconstituted tobacco" refers to a portion of tobacco filler made from tobacco dust and other tobacco scrap materials, processed into sheets, and cut into strips to resemble tobacco. In addition to cost reduction, reconstituted tobacco is very important for its contribution to cigarette taste through processed flavor development using a reaction between ammonia and sugars. In one aspect, a tobacco product comprises reconstituted tobacco.

[0215] As used herein, "expanded tobacco" refers to a portion of tobacco filler that has been treated with a suitable gas expansion, resulting in the tobacco being "puffed" and resulting in reduced density and greater filling capacity. This reduces the weight of tobacco used in a cigarette. In one aspect, a tobacco product comprises expanded tobacco.

[0216] Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly smoking articles containing filter elements, in which a rod of smokable material contains dried tobacco in a tobacco blend. In one aspect, the tobacco product of the present disclosure is selected from the group consisting of cigarillos, non-breathable recess filter cigarettes, breathable recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarettes, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco. In another aspect, the tobacco product of the present disclosure is a smokeless tobacco product. Smokeless tobacco products are not combusted and include, but are not limited to, chewing tobacco, moist smokeless tobacco, snus, and dry snuff. Chewing tobacco is coarsely divided tobacco leaves, which are usually packaged in large bags and used as plugs or twists. Moist smokeless tobacco is moist, more finely divided tobacco, which is provided in a loose or pouched form, usually contained in a round can, and used as a pouch or pinch placed between the cheek and gum of an adult tobacco consumer. Snus is smokeless tobacco that has been heat-treated. Dry snuff is finely ground tobacco that is placed in the mouth or used in the nose. In yet another aspect, the tobacco product of the present disclosure is selected from the group consisting of an electronically heated cigarette, an electronic cigarette, and an electronic vaporizer.

[0217] In one aspect, the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

[0218] In one aspect, the tobacco product of the present disclosure can be a blended tobacco product.

[0219] In another aspect, the present disclosure provides a tobacco blend comprising a dried tobacco material. The tobacco blend can include any combination of dried tobacco, never-cured tobacco, fermented tobacco, unfermented tobacco, expanded tobacco, and reconstituted tobacco.

[0220] In one aspect, the tobacco blend comprises at least 5% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 10% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 15% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 20% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 25% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 30% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 35% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 40% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 45% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 50% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 55% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 60% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 65% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 70% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 75% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 80% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 85% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 90% by weight of cured tobacco. In one aspect, the tobacco blend comprises at least 95% by weight of cured tobacco.

[0221] In one aspect, the tobacco blend comprises at least 5% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 10% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 15% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 20% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 25% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 30% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 35% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 40% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 45% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 50% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 55% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 60% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 65% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 70% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 75% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 80% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 85% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 90% by volume of cured tobacco. In one aspect, the tobacco blend comprises at least 95% by volume of cured tobacco.

[0222] array The term "percent identity" or "percent identical," as used herein with respect to two or more nucleotide or amino acid sequences, is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a comparison window (an "alignable" region), (ii) determining the number of positions where the same nucleic acid base (in the case of nucleotide sequences) or amino acid residue (in the case of proteins and polypeptides) is present in both sequences to calculate the number of matching positions, (iii) dividing the number of matching positions by the total number of positions in the comparison window, and then (iv) multiplying this quotient by 100% to calculate the percent identity. When "percent identity" is calculated with respect to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matching positions over the alignment region by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing for gaps in their alignment), the "percent identity" with respect to a query sequence equals the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), multiplied by 100%.

[0223] When using percentage sequence identity for amino acids, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0224] For optimal alignment of sequences to calculate percent identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), which can be used to compare sequence identity and similarity between two or more nucleotide or amino acid sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the above-mentioned percent identity ranges) can be as determined by the ClustalW algorithm, e.g., Chenna et al., "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research 31: 3497-3500 (2003); Thompson et al., "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal X version 2.0," Bioinformatics 23: 2947-48 (2007); and Altschul et al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the contents and disclosure of which are incorporated herein by reference in their entirety.

[0225] The term "percent complementarity" or "percent complementary" as used herein in reference to two nucleotide sequences is similar to the concept of percent identity, but refers to the percentage of nucleotides in a query sequence that optimally base-pair or hybridize with nucleotides in a subject sequence when the query and subject sequences are linearly aligned and optimally base-paired without secondary folded structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended configuration (i.e., without folded or secondary structures) over the comparison window, (ii) determining the number of base-paired positions between the two sequences over the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined based on the known pairing of nucleotide bases, such as GC, AT, and AU, by hydrogen bonding.When calculating "percent complementarity" relative to a reference sequence without specifying a specific comparison window, the percent identity is determined by dividing the number of complementary positions between two linear sequences by the total length of the reference sequence.Therefore, for the purposes of this application, when two sequences (query and subject) are optimally base-paired (allowing for mismatched or non-base-paired nucleotides), the "percent complementarity" to the query sequence is equal to the number of base-paired positions between two sequences divided by the total number of positions in the query sequence over its length, multiplied by 100%.

[0226] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides containing deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules can contain ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure encompass all forms of sequences, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In another aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single-stranded. In another aspect, the nucleic acid molecules provided herein are double-stranded. The nucleic acid molecules can encode polypeptides or small RNA molecules.

[0227] As used herein, "recombinant nucleic acid" refers to a nucleic acid molecule formed by laboratory methods of genetic recombination, including, but not limited to, molecular cloning.

[0228] Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method, including, but not limited to, recombinant nucleic acid technology and / or polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized as single nucleic acid molecules or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods, such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified by, for example, expressing nucleic acids in expression vectors. Furthermore, purified polypeptides can be obtained by chemical synthesis. The degree of purity of a polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0229] In one aspect, the present disclosure provides a method for detecting recombinant nucleic acids and polypeptides in plant cells.Nucleic acids can also be detected by hybridization, but not limited to this.Nucleic acid hybridization is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0230] In one aspect, the nucleic acid sequences provided herein are at least 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 96% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49. In one aspect, the nucleic acid sequences provided herein are at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48, and 49.In one aspect, the nucleic acid sequences provided herein are 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-27, 34-42, 44, 45, 48 and 49.

[0231] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein. A protein provided herein can be encoded by a nucleic acid molecule provided herein. A protein can include a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell.

[0232] Polypeptides can be detected using antibodies.Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation and immunofluorescence.The antibodies provided herein can be polyclonal or monoclonal antibodies.Antibodies with specific binding affinity for the polypeptides provided herein can be produced using methods well known in the art.The antibodies provided herein can be attached to solid supports such as microtiter plates using methods well known in the art.

[0233] Detection (e.g., of amplification products, hybridization complexes, or polypeptides) can be achieved using a detectable label. The term "label" is intended to encompass the use of direct as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

[0234] In one aspect, the amino acid sequences provided herein are at least 70% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 75% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 80% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 85% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 90% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 95% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 96% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 97% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 98% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50. In one aspect, the amino acid sequences provided herein are at least 99% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47, and 50.In one aspect, the amino acid sequences provided herein are 100% identical or similar to a sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47 and 50.

[0235] The following exemplary, non-limiting embodiments are contemplated: 1. A modified tobacco plant or part thereof comprising a non-naturally occurring mutation in a polynucleotide having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18, 16, 17, 19-21, and 48, A modified tobacco plant or part thereof, wherein the modified tobacco plant comprises a buff phenotype, and wherein the mutation is relative to a control tobacco plant of the same tobacco variety. 2. The modified tobacco plant or part thereof of embodiment 1, wherein the modified tobacco plant is of a tobacco variety selected from the group consisting of flue-cured, bright, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish varieties. 3. The modified tobacco plant or part thereof of embodiment 1 or 2, wherein the modified tobacco plant is of a variety selected from the group consisting of the tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. 4. The modified tobacco plant or part thereof of any one of aspects 1 to 3, wherein the plant is heterozygous for the mutation. 5. The modified tobacco plant or part thereof of any one of aspects 1 to 3, wherein the plant is homozygous for the mutation. 6. The modified tobacco plant or part thereof of any one of embodiments 1-5, wherein the modified tobacco plant is a hybrid. 7. The modified tobacco plant or part thereof of any one of aspects 1-6, wherein the modified tobacco plant is male sterile or cytoplasmic male sterile. 8. The modified tobacco plant or part thereof of any one of aspects 1-7, wherein the non-naturally occurring mutation results in a reduced expression level of the gene compared to a control tobacco plant. 9. The modified tobacco plant or part thereof of any one of embodiments 1-8, wherein the non-naturally occurring mutation results in a reduced level of activity by a protein or polypeptide encoded by the polynucleotide comprising the non-naturally occurring mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-naturally occurring mutation. 10. The modified tobacco plant or part thereof of any one of aspects 1-7, wherein the non-naturally occurring mutation results in an increased level of expression of the gene compared to a control tobacco plant. 11. The modified tobacco plant or part thereof of any one of embodiments 1-7 or 10, wherein the non-naturally occurring mutation results in an increased level of activity by a protein or polypeptide encoded by the polynucleotide comprising the non-naturally occurring mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-naturally occurring mutation. 12. The modified tobacco plant or part thereof of any one of embodiments 1-11, wherein the non-naturally occurring mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5' UTR, an intron, an exon, a 3' UTR, a terminator, and any combination thereof. 13. The modified tobacco plant or part thereof of any one of embodiments 1-12, wherein the non-naturally occurring mutation 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. 14. The modified tobacco plant or part thereof of any one of embodiments 1-13, wherein the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to a wild-type gene selected from the group consisting of SEQ ID NOs: 18, 16, 17, 19-21, and 48. 15. The modified tobacco plant or part thereof of any one of aspects 1-14, wherein the tobacco plant comprises a mutation or transgene that confers reduced nicotine levels. 16. The modified tobacco plant or part thereof of embodiment 15, wherein the tobacco plant is a low-alkaloid tobacco plant. 17. The modified tobacco plant or part thereof of embodiment 15, wherein the mutation that confers reduced nicotine levels comprises a nic1 mutation, a nic2 mutation, or both. 18. The modified tobacco plant or part thereof of embodiment 17, wherein the nic1 mutation, the nic2 mutation, or both, are introgressed from or derived from a variety selected from the group consisting of LA Burley 21, LAFC53, LN B&W, and LN KY171. 19. The modified tobacco plant, or part thereof, of embodiment 15, wherein the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, an ethylene response factor (ERF) transcription factor, a nicotine uptake permease (NUP), and a MATE transporter. 20. The modified tobacco plant or part thereof of embodiment 15, wherein the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus encoding a protein selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. 21. The modified tobacco plant or part thereof of embodiment 15, wherein the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. 22. The modified tobacco plant, or part thereof, of embodiment 15, wherein the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, an ethylene response factor (ERF) transcription factor, a nicotine uptake permease (NUP), and a MATE transporter. 23. The modified tobacco plant or part thereof of embodiment 15, wherein the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. 24. The modified tobacco plant or part thereof of embodiment 15, wherein the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. 25. A modified tobacco plant or part thereof comprising a recombinant nucleic acid construct comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule, The modified tobacco plant or part thereof, wherein the non-coding RNA molecule is capable of binding to RNA encoding an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47 and 50, the non-coding RNA molecule suppresses expression of the amino acid sequence, and the modified tobacco plant comprises a pale yellow phenotype. 26. The modified tobacco plant or part thereof of embodiment 25, wherein the non-coding RNA molecule is a microRNA molecule. 27. A cured tobacco material from the tobacco plant of any one of aspects 1-25. 28. The cured tobacco material of embodiment 27, produced by a drying process selected from the group consisting of hot air drying, air drying, flame drying, and sun drying. 29. A tobacco blend comprising the dried tobacco material of embodiment 27 or 28. 30. The tobacco blend of embodiment 29, wherein the dried tobacco material constitutes at least 10% by weight of the dried tobacco in the tobacco blend. 31. The tobacco blend of embodiment 29, wherein the dried tobacco material constitutes at least 10% by volume of the dried tobacco in the tobacco blend. 32. A tobacco product comprising the dried tobacco material of embodiment 27 or 28. 33. The tobacco product of embodiment 32, wherein the tobacco product is selected from the group consisting of cigarillos, non-breathable recess filter cigarettes, breathable recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarettes, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco. 34. The tobacco product of embodiment 32, wherein the tobacco product is a smokeless tobacco product. 35. The tobacco product of embodiment 32, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus. 36. A reconstituted tobacco comprising the dried tobacco material of embodiment 27 or 28. 37. A method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, comprising: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and linked within 20 centimorgans (cM) of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising the PY QTL and the one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting a pale yellow phenotype. The method comprising: 38. A method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, comprising: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and located within 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising the PY QTL and the one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting a pale yellow phenotype. The method comprising: 39. Crossing the one or more tobacco plants produced in step (c) with a tobacco plant containing a mutation or transgene that directly suppresses or eliminates the expression or activity of one or more genes encoding products selected from the group consisting of methylputrescine oxidase (MPO), quinolate phosphoribosyltransferase (QPT), quinolinate synthase (QS), BBL, A622, aspartate oxidase, agmatine deiminase (AIC), S-adenosyl-methionine synthetase (SAMS), arginase, diamine oxidase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), arginine decarboxylase (ADC), nicotine uptake permease (NUP), and MATE transporter. 39. The method of embodiment 37 or 38, further comprising: 40. The method of embodiment 37, wherein the one or more marker loci are linked within 15 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. 41. The method of embodiment 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 10 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. 42. The method of embodiment 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. 43. The method of embodiment 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 1 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. 44. The method of embodiment 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 0.5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5. 45. The method of embodiment 38, wherein the one or more marker loci are located within 500,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. 46. ​​The method of embodiment 38, wherein the one or more marker loci are located within 250,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. 47. The method of embodiment 38, wherein the one or more marker loci are located within 100,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. 48. The method of embodiment 38, wherein the one or more marker loci are located within 50,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5. 49. The method of embodiment 37 or 38, wherein the buff phenotype comprises yellowing of at least 25% of the leaves of the progeny plant, and on average at least 80%. 50. The method of any one of aspects 37-49, wherein the locus is SEQ ID NO: 1. 51. The method of any one of aspects 37-49, wherein the locus is SEQ ID NO: 2. 52. The method of any one of aspects 37-49, wherein the locus is SEQ ID NO: 3. 53. The method of any one of aspects 37-49, wherein the locus is SEQ ID NO: 4. 54. The method of any one of aspects 37-49, wherein the locus is SEQ ID NO: 5. 55. The method of any one of embodiments 37-49, wherein the genotyping in step (a) comprises detecting one or more marker loci. 56. The method of embodiment 55, wherein the one or more marker loci are selected from the group consisting of one or more single nucleotide polymorphism (SNP) markers, one or more insertion-deletion (INDEL) markers, one or more simple sequence repeat (SSR) markers, one or more restriction fragment length polymorphism (RFLP) markers, one or more random amplified polymorphic DNA (RAPD) markers, and one or more amplified fragment length polymorphism (AFLP) markers. 57. The method of embodiment 55, wherein genotyping comprises the use of oligonucleotide probes. 58. The method of embodiment 57, wherein the oligonucleotide probe comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42. 59. The method of embodiment 57, wherein the oligonucleotide probes flank polymorphic nucleotide positions of the marker locus. 60. The method of any one of embodiments 37-49, wherein the genotyping in step (a) comprises detecting haplotypes. 61. The method of embodiment 60, wherein the haplotype comprises at least two SNPs selected from the group consisting of a guanine at nucleotide 121 of SEQ ID NO: 1, a guanine at nucleotide 121 of SEQ ID NO: 2, a guanine at nucleotide 101 of SEQ ID NO: 3, a thymine at nucleotide 121 of SEQ ID NO: 4, and a guanine at nucleotide 121 of SEQ ID NO: 5. 62. The method of any one of aspects 37-61, wherein the tobacco plant is of a tobacco variety selected from the group consisting of Flue-Cured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish. 63. The method of any one of embodiments 37-61, wherein the tobacco plant is of a variety selected from the group consisting of tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. 64. The method of embodiment 37 or 38, wherein the one or more marker loci are selected from the group consisting of SEQ ID NOs: 1-5. 65. One or more marker loci are: (a) guanine at nucleotide position 121 of SEQ ID NO: 1; (b) a guanine at nucleotide position 121 of SEQ ID NO: 2; (c) a guanine at nucleotide position 101 of SEQ ID NO: 3; (d) a thymine at nucleotide 121 of SEQ ID NO: 4; and (e) guanine at nucleotide 121 of SEQ ID NO: 5 65. The method of embodiment 64, wherein the single nucleotide polymorphism is selected from the group consisting of: 66. A method for introgressing pale yellow (PY) QTL, comprising: (a) crossing a first tobacco plant containing the PY quantitative trait locus (QTL) with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) (i) a guanine at nucleotide 121 of SEQ ID NO: 1; (ii) a guanine at nucleotide 121 of SEQ ID NO: 2; (iii) a guanine at nucleotide 101 of SEQ ID NO: 3; (iv) a thymine at nucleotide 121 of SEQ ID NO: 4, or (v) guanine at nucleotide 121 of SEQ ID NO: 5 selecting a progeny plant or seed produced in step (a) that comprises at least one PY-associated single nucleotide polymorphism (SNP) selected from the group consisting of: The method comprising: 67. A method for introgressing a pale yellow (PY) trait, comprising: (a) crossing a first tobacco plant containing a non-naturally occurring mutation in a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48 with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) selecting progeny plants or seeds produced in step (a) that contain the non-naturally occurring mutation, wherein the progeny plants or seeds contain the pale yellow trait. The method comprising: 68. The method of embodiment 66, wherein the progeny plant or seed of step (b) is heterozygous for the SNP. 69. The method of embodiment 66, wherein the progeny plants or seeds of step (b) are homozygous for the SNP. 70. The method of any one of embodiments 66-69, wherein the progeny plant or seed of step (b) comprises at least two SNPs selected from the group consisting of a guanine at nucleotide 121 of SEQ ID NO: 1, a guanine at nucleotide 121 of SEQ ID NO: 2, a guanine at nucleotide 101 of SEQ ID NO: 3, a thymine at nucleotide 121 of SEQ ID NO: 4, and a guanine at nucleotide 121 of SEQ ID NO: 5. 71. The method of any one of embodiments 66-69, wherein the single nucleotide polymorphism is a guanine at nucleotide 121 of SEQ ID NO: 1. 72. The method of any one of embodiments 66-69, wherein the single nucleotide polymorphism is a guanine at nucleotide 121 of SEQ ID NO: 2. 73. The method of any one of embodiments 66-69, wherein the single nucleotide polymorphism is a guanine at nucleotide 101 of SEQ ID NO: 3. 74. The method of any one of embodiments 66-69, wherein the single nucleotide polymorphism is a thymine at nucleotide 121 of SEQ ID NO: 4. 75. The method of any one of embodiments 66-69, wherein the single nucleotide polymorphism is a guanine at nucleotide 121 of SEQ ID NO: 5. 76. The method of any one of embodiments 66-75, wherein the tobacco plant is of a tobacco variety selected from the group consisting of Flue-Cured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish. 77. The method of any one of embodiments 66-75, wherein the tobacco plant is of a variety selected from the group of tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8. 78. The method of embodiment 67, wherein the progeny plant or seed is heterozygous for the non-naturally occurring mutation. 79. The method of embodiment 67, wherein the progeny plant or seed is homozygous for the non-naturally occurring mutation. 80. The method of embodiment 67, wherein the non-naturally occurring mutation results in a reduced expression level of the gene compared to a control tobacco plant. 81. The method of embodiment 67, wherein the non-naturally occurring mutation results in an increased expression level of the gene compared to a control tobacco plant. 82. The method of embodiment 67, wherein the non-naturally occurring mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5' UTR, an intron, an exon, a 3' UTR, a terminator, and any combination thereof. 83. The method of embodiment 67, wherein the non-naturally occurring mutation 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. 84. The method of embodiment 67, wherein the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to a wild-type nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48. 85. The method of embodiment 66, wherein the progeny plant or seed comprises a reduced level of at least one tobacco-specific nitrosamine (TSNA) compared to the second tobacco plant lacking the PY QTL. 86. The method of embodiment 85, wherein the at least one TSNA is selected from the group consisting of N-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone, N'-nitrosoanatabine, and N'-nitrosoanabasine. 87. The method of embodiment 66, wherein the progeny plants or seeds comprise an increased USDA leaf grade index compared to the second tobacco plant lacking the PY QTL.

[0236] Having now generally described the present disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the disclosure unless otherwise specified. [Example]

[0237] Example 1. Mapping the pale yellow (PY) locus in tobacco The Pale Yellow (PY) locus is known to accelerate senescence in tobacco, however, the location of the PY locus within the tobacco genome is unknown.

[0238] To determine the location of the PY locus, an F2 mapping population was generated from a cross between narrow-leaf Madole LC (NL Madole LC, PY-deficient) and TI1372 (the origin of the PY trait). The F2 individuals were scored phenotypically in the field by visual observation of the ripening process after topping and by visually scoring the leaves of each plant before and after treatment with ethephon (see Tables 9-12). Leaves were removed from the plants and immersed in an ethephon treatment solution (1.2 g ai / L) to thoroughly wet them.

[0239] Leaves are screened each of five consecutive days after ethephon treatment. Ethephon is converted to ethylene within the plant, which induces ripening, and plants carrying the PY trait exhibit more rapid or accelerated yellowing upon ethephon application.

[0240] Table 9. Phenotypic scoring scale for untreated tobacco leaves TIFF2026035667000037.tif25150

[0241] Table 10. Phenotypic scoring scale for ethephon-treated tobacco leaves TIFF2026035667000038.tif29150

[0242] Table 11. Phenotypic scoring of F2 mapping individuals Tables 9 and 10 provide an explanation of the scoring used in this table. TIFF2026035667000039.tif121129TIFF2026035667000040.tif228129TIFF2026035667000041.tif22812 9TIFF2026035667000042.tif228129TIFF2026035667000043.tif228129TIFF2026035667000044.tif50129

[0243] Table 12. Phenotypic scoring of control plants Table 10 provides an explanation of the scoring used in this table. All control plants were NL Madole LC plants. TIFF2026035667000045.tif114128

[0244] Ninety-three of the F2 individuals scored in Table 12, as well as the parental lines (NL Madol LC and TI1372), were genotyped using a custom tobacco axiom array containing approximately 170,000 SNPs distributed across the entire tobacco genome. Genotypic and phenotypic data were combined to identify quantitative trait loci and the genetic control of the pale yellow color trait. A tobacco pseudomolecule QTL on chromosome 15 (PY QTL) explained 75% of the PY trait variability identified in this analysis. The PY QTL was located between the published SSR / microsatellite markers PT51549 and PT55414. See Bindler et al., "A high-density genetic map of tobacco (Nicotiana tabacum L.) obtained from large-scale microstatellite marker development," Theor. Appl. Genet., 123:219-230 (2011), which is incorporated herein by reference in its entirety. Additional published tobacco markers can be found in Tong et al., "Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco," Breed Sci., 66:381-390 (2016), which is also incorporated herein by reference in its entirety.

[0245] Example 2. Design and validation of a high-throughput KASP™ assay KASP™ is a competitive allele-specific PCR-based genotyping assay system that allows biallelic scoring of SNPs at specific loci. KASP™ primers have been designed for five SNP markers within the QTL identified in Example 1. See Table 13.

[0246] Table 13. SNPs within PY QTL assayed using KASP™ The SNP position is shown in the sequence column of the table. Bold Underline is. TIFF2026035667000046.tif101163TIFF2026035667000047.tif207163TIFF2026035667000048.tif159163

[0247] The 80 F2 individuals phenotyped in Example 1 are screened at the SNP loci identified above in Table 13 to identify SNP markers associated with the PY trait. See Table 14.

[0248] Table 14. KASP™ SNP Marker Results The genotype column indicates the nucleotide observed at the SNP position for each PY_SNP marker, as described above in Table 13. "?" indicates that the data was inconclusive. "WT" refers to the wild-type or non-PY genotype. TIFF2026035667000049.tif34163TIFF2026035667000050.tif228163TIFF2026035667000051.tif157163

[0249] Table 15 provides the physical locations of the SNP markers identified herein relative to the published markers PT51549 (GenBank Accession No.; Pr032530969), PT55414 (GenBank Accession No. Pr032533458), PT50034 (GenBank Accession No. Pr032529969), and PT53131 (GenBank Accession No. Pr032531997).

[0250] Table 15. Physical locations of markers and candidate genes on chromosome 15 of the tobacco genome. TIFF2026035667000052.tif90150

[0251] Example 3. Identification of candidate genes involved in PY traits Dark tobacco varieties KY171 (lacking the PY trait) and PYKY171 (KY171 containing the PY trait introgressed from TI1372), as well as TI1372, were grown in a greenhouse until the plants reached flowering. After flowering, all plants were pinched, and leaf samples were collected from the plants 24 hours, 48 ​​hours, 72 hours, 1 week, and 2 weeks after pinching.

[0252] RNA is collected from all topped samples per cultivar at each time point, as well as from untopped control samples, and subjected to 2x100 nucleotide end sequencing on an Illumina HiSeq at a depth of approximately 60,000,000 paired-end reads per sample.

[0253] Based on the RNAseq results, six candidate genes have been identified on chromosome 15. See Table 16. g61524 does not fall within the PY QTL, but showed significantly altered expression between the two tobacco varieties tested.

[0254] Table 16: Candidate genes TIFF2026035667000053.tif162161

[0255] Example 4. Validation of identified candidate genes Plants from the same seed lot scored in the F2 mapping population (see Example 1) above are used to track the expression of the candidate genes provided in Example 3 above. The parental lines are used as controls. Gene expression is examined in plants scored as homozygous PY, heterozygous PY, and WT, as determined by the KASP™ SNP marker results (see Table 14). Approximately 24 F2 individuals (homozygous PY, heterozygous PY, and WT) per group are screened for candidate gene expression.

[0256] Expression of candidate genes is measured in leaf tissue using quantitative RT-PCR (qPCR), TaqMan™ gene expression assay, and RNAseq. Leaf tissue is collected from each F2 plant before and at several time points after pruning (e.g., 24 hours, 48 ​​hours, 72 hours, 1 week, and 2 weeks after pruning). The same lines as those used in the RNAseq study in Example 3 are used here.

[0257] TaqMan™ Gene Expression Assays (see Table 17 for primers and probes) are designed to monitor the expression of the candidate genes identified in Example 3 above.

[0258] Using qPCR, it was shown that expression of g61524 and g58917 was similar in KY171, Pale Yellow KY171, and TI1372. See Figures 2 and 3. However, expression of g58899 was significantly lower in Pale Yellow KY171 and TI1372 compared to KY171. See Figure 1.

[0259] Table 17. TaqMan™ primers and probes TIFF2026035667000054.tif103160

[0260] Example 5. Transformation and regeneration of modified tobacco plants to overexpress candidate genes Candidate genes that show increased expression correlated with the PY trait (e.g., TI1372, F2 individuals homozygous for the PY trait) are overexpressed in a non-PY background (e.g., NL Madole NC) and the resulting phenotype observed.

[0261] Using an expression vector as a backbone, multiple transformation vectors containing recombinant DNA constructs that overexpress candidate genes (e.g., nucleic acid sequences encoding any one of SEQ ID NOs: 28-33, 46, 47, and 50; see also SEQ ID NOs: 53 and 54) are generated. The expression vectors contain a cassette containing a CaMV 35S promoter, a NOS terminator, and a kanamycin selectable marker (NPT II) operably linked to the actin 2 promoter and a NOS terminator. The nucleic acid vector containing the transgene of interest is introduced into tobacco leaf discs by Agrobacterium transformation. See, e.g., Mayo et al., 2006, Nat Protoc. 1:1105-11 and Horsch et al., 1985, Science 227:1229-1231.

[0262] NL Madole LC tobacco plants were grown in Magenta™ GA-7 boxes, and leaf discs were cut and placed in Petri dishes. Agrobacterium tumefaciens cells containing the transformation vector were collected by centrifuging 20 mL of cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10 minutes. The supernatant was removed, and the Agrobacterium tumefaciens cell pellet was resuspended in 40 mL of liquid resuspension medium. Tobacco leaves were cut into eight 0.6 cm discs with a No. 15 razor blade, avoiding the midrib, and placed upside down in a Petri dish. A thin layer of Murashige & Skoog (MS) B5 Vitamin Liquid Resuspension Medium was added to the Petri dish, and the leaf discs were evenly pierced with a fine-tipped needle. Add approximately 25 mL of the Agrobacterium tumefaciens suspension to the Petri dish and incubate the leaf discs in the suspension for 10 min.

[0263] Transfer the leaf discs to a co-cultivation Petri dish (1 / 2 MS medium) and place the discs upside down in contact with a filter paper layered with co-cultivation TOM medium (MS medium with 30 g / L sucrose; 0.1 mg / L 1-naphthaleneacetic acid (NAA); and 1 mg / L 6-benzylaminopurine (BAP)). Seal the Petri dish with parafilm before incubation in the dark for 2 days.

[0264] After incubation, leaf discs were transferred to regeneration / selection TOM-Hyg medium Petri dishes (TOM medium supplemented with 200 mg / L cefotaxime and 50 mg / L hygromycin). Calli formed from the leaf discs were subcultured biweekly onto fresh TOM-Hyg medium in dim light (60 mE / ms to 80 mE / ms) and maintained at 24°C under an 18-hour light, 6-hour dark photoperiod until shoots (plantlets) could be excised. Plantlets formed from the callus were removed with forceps and subcultured onto MS rooting medium (MS medium with 3 g / L sucrose; 7 g / L dextrose; 200 mg / L cefotaxime; 50 mg / L hygromycin). Shoots on MS rooting medium were incubated at 24°C under dim light and an 18-hour light, 6-hour dark photoperiod to induce rooting.

[0265] Once the plantlets, including both shoots and roots, are large enough (e.g., more than half the height of a Magenta™ GA-7 box), they are transferred to Jiffy peat pellets for acclimation in a growth room. Once established, the seedlings are transferred to a greenhouse for further growth, breeding, and analysis.

[0266] The resulting plants are evaluated by visual observation or ethephon screening as described in Example 1 above.

[0267] 4 shows the results of semi-quantitative RT-PCR (using RNA extracted from young leaves) of 13 TO tobacco lines overexpressing g58899 (SEQ ID NO:24, encoding SEQ ID NO:30). Some lines (e.g., lines 3 and 11) show silencing of expression compared to wild-type (WT) controls, while other lines (e.g., lines 9 and 12) show increased expression. Without being limited by any scientific theory, it is known in the art that the insertion of multiple copies of a single transgene can lead to gene silencing.

[0268] The TO lines tested in Figure 4 were examined phenotypically. Lines 9 and 12, which were shown to overexpress g58899, exhibit green leaves throughout. See Figure 5. In contrast, lines 3 and 11, which were shown to have reduced expression of g58899, exhibit a PY phenotype in older leaves. See Figure 5, black arrows.

[0269] Example 6. Knockdown of candidate genes Candidate genes that show reduced expression correlated with the PY trait (e.g., TI1372, F2 individuals homozygous for the PY trait) are targets for knocking down or knocking out expression in a non-PY background (e.g., NL Madole NC).

[0270] An artificial miRNA or other RNAi construct is created to produce an miRNA or another non-coding RNA capable of reducing the expression of each of the candidate genes (e.g., SEQ ID NOs: 22-27). The artificial miRNA or RNAi construct is inserted into a plasmid under the control of the CaMV 35S promoter. The plasmid further contains a cassette containing a NOS terminator and a kanamycin selectable marker (NPT II) operably linked to the actin 2 promoter and NOS terminator.

[0271] Tobacco leaf discs are transformed and tobacco plants are regenerated as described in Example 5.

[0272] Plants containing a construct designed to suppress g58899 (SEQ ID NOs: 51 and 52) began to exhibit a PY phenotype in some leaves before removal from the Magenta™ GA-7 box ( Figure 6 ; see particularly the black arrowheads). This effect was not observed in plants expressing the g58905 (SEQ ID NO: 26) RNAi suppression construct or in wild-type controls.

[0273] Seventeen transgenic lines containing the g58899 suppression construct are examined for g58899 expression levels using RNA extracted from young plants. See Figure 7. Figure 8 shows the phenotypes observed in plants containing various levels of g58899 suppression compared to wild-type (WT) controls. All plants showing suppression of g58899 compared to wild-type controls exhibit the PY phenotype in older leaves.

[0274] Example 7. Mutations in candidate genes Candidate genes that show reduced expression correlated with the PY trait (e.g., TI1372, F2 individuals homozygous for the PY trait) are targets for knocking down or knocking out expression in a non-PY background (e.g., NL Madole NC).

[0275] Mutations are generated in the genomic sequence of each of the candidate genes (e.g., SEQ ID NOs: 16-21 and 48). Tobacco protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding CRISPR proteins or CRISPR proteins and specific guide RNAs (gRNAs) targeting individual genes at the desired locations.

[0276] The transfected protoplasts are then immobilized on 1% agarose beads and subjected to tissue culture. When the calli grow to approximately 1 millimeter in diameter, they are spread onto TOM2 plates. The calli are screened for mutations (e.g., insertions or deletions (IDs)) at the target location using fragment analysis. Candidates that show a change in size compared to the wild-type control are selected for further culture, and the resulting shoots are again tested by fragment analysis to confirm the presence of the mutation.

[0277] The resulting plants are evaluated by visual observation or ethephon screening as described in Example 1 above.

[0278] Example 7. Leaf quality of cultivars containing the Pale Yellow trait Leaf quality values ​​were obtained from flue-cured (Table 18), burley (Table 19), and dark (Table 20) tobacco varieties at a field site in Virginia, USA, in 2018. Each trial included three or four replicates in a randomized complete block design. Grading was performed after curing was completed. Each replicate was assigned a USDA grade index by a certified tobacco leaf grading judge. Grading was performed separately for several leaf positions (A through D). At harvest, leaves were divided into three-leaf positions. The top three leaves (e.g., the youngest leaves) were designated position "A," leaves 4–6 were designated position "B," leaves 7–9 were designated position "C," leaves 10–12 were designated position "D," leaves 13–15 were designated position "E," and so on until the last leaf.

[0279] Table 18: Leaf Grade Index Values ​​for Flue-Cured Tobacco PY refers to varieties containing the Pale Yellow trait. LA refers to varieties containing low alkaloid levels. TIFF2026035667000055.tif100150

[0280] Table 19: Burley Tobacco Leaf Grade Index PY refers to varieties containing the Pale Yellow trait. LA refers to varieties containing low alkaloid levels. TIFF2026035667000056.tif96150

[0281] Table 20: Dark Tobacco Leaf Grade Index PY refers to varieties containing the Pale Yellow trait. LN refers to varieties containing low nicotine levels. NG means no rating was given. TIFF2026035667000057.tif148135

[0282] Example 8. Further validation of identified candidate genes The expression of candidate genes g58899, g58917, and g61524 was further examined in tobacco cultivars Narrow Leaf Madole (NLM; lacking the pale yellow trait), ds1771 (an F2 population generated by crossing NLM with TI1372), wild type (WT; without the pale yellow phenotype), ds1771 (HT) heterozygous for the pale yellow trait, ds1771 (HM) homozygous for the pale yellow trait, and TI1372 (source of the pale yellow trait) before (UT) and 24 hours after (24 hours) pinching.

[0283] The ds1771 line was genotyped using the SNP markers identified in Table 13 and expression analysis was performed as described in Example 4 before topping and at 24 hours after topping.

[0284] Expression of g58899 was significantly lower in cultivars ds1771-HT, ds1771-HM, and TI1372 compared to NLM and ds1771-WT (see Figure 9). Expression levels of g58917 and g61524 were similar in all lines (see Figures 10 and 11, respectively).

[0285] Example 9. Further sequence analysis of g58899 Gene g58899 shows 87.77% sequence identity with the tomato (Solanum lycopersicum) STAY-GREEN gene (GenBank accession number NP_001358338.1; SEQ ID NO: 43). See Table 16. In tomato, the STAY-GREEN protein is required to trigger chlorophyll breakdown during leaf senescence and fruit ripening.

[0286] Sequencing of the cDNA sequences of g58899 (SEQ ID NO: 44) in Narrow Leaf Madole, which lacks the PY trait, and TI1372 (SEQ ID NO: 45), which contains the PY trait, shows that these sequences share 100% identity at the nucleotide level. Therefore, the amino acids encoded by SEQ ID NOs: 44 and 45 also share 100% identity at the protein level (SEQ ID NOs: 46 and 47, respectively).

[0287] Alignment of the TI1372 cDNA (SEQ ID NO: 45) to the internal tobacco genome assembly shows that the best match is g58899. Similarly, alignment of the Narrow Leaf Madole cDNA (SEQ ID NO: 44) to the internal tobacco genome assembly shows that the best match is also g58899.

[0288] Alignment of the TN90 cultivar g58899 amino acid sequence (SEQ ID NO: 30) with the Arabidopsis proteins BALANCE OF CHLOROPHYLL METABOLISM1 (BCM1) and BCM2 demonstrates that g58899 shares 66.21% identity with BCM1 (SEQ ID NO: 55) and 64.13% identity with BCM2 (SEQ ID NO: 56). See Figure 12. BCM1 and BCM2 have been shown to be involved in the suppression or attenuation of chlorophyll degradation. See Wang et al., Nature Communications, 11:1254 (2020).

[0289] Alignment of the NLM g58899 amino acid sequence (SEQ ID NO: 46), the TN90 g58899 amino acid sequence (SEQ ID NO: 30), and the K326 g58899 amino acid sequence (SEQ ID NO: 57) allows the identification of the g58899 consensus amino acid sequence (SEQ ID NO: 58). See Figure 13.

[0290] Example 10. Expression profile of g58899 The expression profile of g58899 (SEQ ID NO: 24) was examined in tobacco lines K326, Pale Yellow (PY) K326, KY 171, Pale Yellow (PY) K171, TN90, and Pale Yellow (PY) TN 90 using qPCR. RNA samples were collected from leaves before and 24 hours after pruning. The expression profiles of g61524 (SEQ ID NO: 27) and g58917 (SEQ ID NO: 25) were also examined. Actin was used as a control. The results are shown in Figures 14A, 14B, and 14C.

[0291] Sequence information SEQUENCE LISTING <110> ALTRIA CLIENT SERVICES LLC <120> PALE YELLOW LOCUS AND ITS APPLICATIONS IN TOBACCO <150> US 62 / 913,414 <151> 2019-10-10 <150> US 62 / 913,313 <151> 2019-10-10 <160> 58 <170> PatentIn version 3.5 <210> 1 <211> 241 <212> DNA <213> Nicotiana tabacum <220> <221> modified_base <222> (121)..(121) <223> a, c, t, g, unknown or other <400> 1 gtcaagcagt ttttgaacaa gttctaccca cccaataaga ttgccaagta agttgatcag 60 atattgagct tcagggagaa tccaactgaa acactacaag aaacgtaaga gaggttcaaa 120 nggatactgg ttaagtgtcc acatcatggt attccagatt agatgttggg gcaaatgttc 180 tacatgggat tgacagacag cttgaaggcc aatgttgatg cttcagcaag tggagcattt 240 t 241 <210> 2 <211> 241 <212> DNA <213> Nicotiana tabacum <220> <221> modified_base <222> (121)..(121) <223> a, c, t, g, unknown or other <400> 2 cttcttctac gcgttcacaa ggtgctggtc acgttcgcga aggtatgagc tggtaaagct 60 ttgcattcgc gaagccgtgg tcgcatttgc gaagggtaag aattgtaaag tttcacgttc 120 ncgaaggatt aaattgtggg caatcgagtt gtgcttcgca aacgcaaggg acctgtcgtg 180 ttcgcgaaga agagaggtca ggacagaagg tttaagttca gaaaatggga cttcgtccca 240 t 241 <210> 3 <211> 201 <212> DNA <213> Nicotiana tabacum <220> <221> modified_base <222> (101)..(101) <223> a, c, t, g, unknown or other <400> 3 gagagcttcg tgctttaagt atggtatcgt ctttgttaga aagtgtttca cgttatatta 60 tggagttgtg caaatctgaa tttagtcggg gcccaatacg nagacaccag gtgggacact 120 aaaaaagaaa agaaaaaaga ggagaaacaa agtccgaagt ctactagata caaatgcata 180 cgtctctatt aataaatttg t 201 <210> 4 <211> 241 <212> DNA <213> Nicotiana tabacum <220> <221> modified_base <222> (121)..(121) <223> a, c, t, g, unknown or other <400> 4 aatagtacaa gatgagagca atttcatata gtcactctca actaattagg aaatatgagg 60 cgcttgactg attgaagttt gtatgttgaa tatactagaa cttctgatgt agacatgtag 120 nattctgtat attttagagc acatcactta taagcagccc aagaatatta ctgtatctaa 180 gacataattt agtaaataaa aagtatgttt tctttgaaag tttaagattt tttatgagat 240 g 241 <210> 5 <211> 241 <212> DNA <213> Nicotiana tabacum <220> <221> modified_base <222> (121)..(121) <223> a, c, t, g, unknown or other <400> 5 tacctcggga gtgccgttgt tgatattttc ctattagtgt acttgtcttg attgttttat 60 ttttccttta atatgtaaat tcctgtttgt cttccgtgat gtattattcg cccttactct nagcagttaa attctgacat actgcttact tgattcactc tcattgttat tattttatta ttattattat ttattattatt ttattattattattattattattattattattattattattattattattattattattattattattattattattattattattattatta a 241 <210> 6 <211> 241 <212> DNA <213> Nicotiana tobacco <400> 6 gtcaagcagt ttttgaacaa gttctaccca cccaataaga ttgccaagta agttgatcag atattgagct tcagggaga tccaactga acactacag aaacgtaga gaggttcaaa aggatactgg ttagtgtcc acatcatggt attccagatt agatgttggg gcaaatgttc 240. tcatgggat tgacagacag cttgaaggcc aatgttgatg cttcagcaag tggagcattt t 241 <210> 7 <211> 241 <212> DNA <213> Nicotiana tobacco <400> 7 cttcttctac gcgttcacaa ggtgctggtc acgttcgcga aggtatgagc tggtaaagct ttgcattcgc gaagccgtgg tcgcatttgc gaagggtaag aattgtaaag tttcacgttc 120 acgaaggatt aaattgtggg caatcgagtt gtgcttcgca aacgcaaggg acctgtcgtg 180 ttcgcgaaga agagaggtca ggacagaagg tttaagttca gaaaatggga cttcgtccca 240 t 241 <210> 8 <211> 201 <212> DNA <213> Nicotiana tabacum <400> 8 gagagcttcg tgctttaagt atggtatcgt ctttgttaga aagtgtttca cgttatatta 60 tggagttgtg caaatctgaa tttagtcggg gcccaatacg aagacaccag gtgggacact 120 aaaaaagaaa agaaaaaaga ggagaaacaa agtccgaagt ctactagata caaatgcata 180 cgtctctatt aataaatttg t 201 <210> 9 <211> 241 <212> DNA <213> Nicotiana tabacum <400> 9 aatagtacaa gatgagagca atttcatata gtcactctca actaattagg aaatatgagg 60 cgcttgactg attgaagttt gtatgttgaa tatactagaa cttctgatgt agacatgtag 120 aattctgtat attttagagc acatcacta taagcagccc aagaatatta ctgtatctaa 180 gacataattt agtaaataaa aagtatgttt tctttgaaag tttaagattt tttatgagat 240 g 241 <210> 10 <211> 241 <212> DNA <213> Nicotiana tabacum <400> 10 tacctcggga gtgccgttgt tgatattttc ctattagtgt acttgtcttg attgttttat 60 ttttccttta atatgtaaat tcctgtttgt cttccgtgat gtattattcg cccttactct 120 aagcagttaa attctgacat actgcttact tgattcactc tcattgttat tattttatta 180 ttattattat tattattat attattatta tattattata tattattat attatatatt 240 a 241 <210> 11 <211> 241 <212> DNA <213> Nicotiana tabacum <400> 11 gtcaagcagt ttttgaacaa gttctaccca cccaataaga ttgccaagta agttgatcag 60 atattgagct tcagggagaa tccaactgaa acactacaag aaacgtaaga gaggttcaaa 120 gggatactgg ttaagtgtcc acatcatggt attccagatt agatgttggg gcaaatgttc 180 tacatgggat tgacagacag cttgaaggcc aatgttgatg cttcagcaag tggagcattt 240 t 241 <210> 12 <211> 241 <212> DNA <213> Nicotiana tabacum <400> 12 cttcttctac gcgttcacaa ggtgctggtc acgttcgcga aggtatgagc tggtaaagct 60 ttgcattcgc gaagccgtgg tcgcatttgc gaagggtaag aattgtaaag tttcacgttc 120 gcgaaggatt aaattgtggg caatcgagtt gtgcttcgca aacgcaaggg acctgtcgtg 180 ttcgcgaaga agagaggtca ggacagaagg tttaagttca gaaaatggga cttcgtccca 240 t 241 <210> 13 <211> 201 <212> DNA <213> Nicotiana tabacum <400> 13 gagagcttcg tgctttaagt atggtatcgt ctttgttaga aagtgtttca cgttatatta 60 tggagttgtg caaatctgaa tttagtcggg gcccaatacg gagacaccag gtgggacact 120 aaaaaagaaa agaaaaaaga ggagaaacaa agtccgaagt ctactagata caaatgcata 180 cgtctcttatt aataaatttg t 201 <210> 14 <211> 241 <212> DNA <213> Nicotiana tabacum <400> 14 aatagtacaa gatgagagca atttcatata gtcactctca actaattagg aaatatgagg 60 cgcttgactg attgaagttt gtatgttgaa tatactagaa cttctgatgt agacatgtag 120 tattctgtat attttagagc acatcactta taagcagccc aagaatatta ctgtatctaa 180 gacataattt agtaaataaa aagtatgttt tctttgaaag tttaagattt tttatgagat 240 g 241 <210> 15 <211> 241 <212> DNA <213> Nicotiana tabacum <400> 15 tacctcggga gtgccgttgt tgatattttc ctattagtgt acttgtcttg attgttttat 60 ttttccttta atatgtaaat tcctgtttgt cttccgtgat gtattattcg cccttactct 120 gagcagttaa attctgacat actgcttact tgattcactc tcattgttat tattttatta 180 ttattattat tattattatt attattatta tattattata tattattatt attatatatt 240 at 241 <210> 16 <211> 1822 <212> DNA <213> Nicotiana tabacum <400> 16 atgcaaacca gagttggtag tagattggtc actgaacagt atcacgatga agaagtacgg 60 ccatatattc aacaattaat gaatgctcag aattgctctc cagcccaaac ttatgataat 120 caatctaata ttttgaacaa ttctgttggt actggagctg agcagaacaa tgaatcaggt 180 tctattttga agttttagta ttgagtccac tattttctta gtattgcttt gcactagctg 240 taggttctgc taaaacaagg agttaattct ccagctcaag actattttct ttgtgaattt 300 gcttgcacta gatcatggac ctttgaatct gtttcaggtc cttcagaggt aagaaaagtt 360 tgtgggccta cactactaaa agatgtttgg aatctaccat caaggaagac aattgatgtg 420 caatagaata gtcgtaatca agctattgga aaaaagggtc gaaagcttgc tagctttcta 480 agtatcattg ctagaacccc agagctgaca ccgttaaata taaatgattg gcgagtattt 540 gacaaagaag aaaagaagaa attggtggag tttgtgaggg tatgcaattt ttatctttaa 600 taagtatcat tctttttata catgatattt tcttgtaaac tcgtgcttta ttttttagaa 660 aaagttctcg attccagtat gtagagaaga gtttataaaaa aagtcaatag ggaaaaaatg 720 gaagactat aaatgtgatt tgaagactat gtatgtgacc aagtataaga gcaaagatgc 780 cttgatgaaa aatagaccaa gtcacatacc aagggatcaa tggactggtc tcgtcttgta 840 ttggctttct gataaagcaa aggtgagtaa gtttttgtgt tgaatctaca tttataact 900 ctctgtcctg ttttcttct ttgaaccttt ttgtgtttat tcaaagaaca tatatttaat 960 aagaatgttt acttggtctt taccagttac atttcaccca cattatacca accaaaatat 1020 ttttagaaag tgctgcaaac gttgtggcag gtggttcaga atatgaattt tgccttttaa 1080 atatagcccc ttgagcacaa tagatgattt ggttattagt ttatctttata tgattaagta 1140 ttgttagtga gtatttttgt gttgcaataa cagaagcgca gtcaagcaaa tagaatcagt 1200 agggctaaac aaaagatgcc tcacacagga ggatccaaaa gcatagcaac cttgatgaat 1260 gaaaaggtat taatgtataa attacatacc ataagattct ataattcttt tctatgatat 1320 ctaattgttt ggcatcaatt taactttatt aggctataga tggaatagag cctacacgtg 1380 ctcaagttta catattaact catacaaagc gtaaggatgg tagaccattg gatgaggaat 1440 cttcaaatac agttgtaaga tttcttataa gttgtatttt ttaagattaa acatatgtac 1500 tagctcatgt ctaatgcatc ataatttgta gaggctttaa atgattgact gatccttaaa 1560 ctttatgtc tcttcttccg gctatttctt ttccattcc atgtctaata tatttccttg 1620 cattaattgc tgatgttaat acaagttatg taagaaactg ccttaacttt gttaactaac 1680 attgacttt tcatatgtgc aaatgctggc tagtttggtt tcacaatatc tcatcttatt 1740 gttagttcgt cctccactgt attgtgtcat aaattgtat ctcaattact gtcaggacat 1800 tgatgaaaga gaagttgagt aa 1822 <210> 17 <211> 911 <212> DNA <213> Nicotiana tabacum <400> 17 atggtagacc attggatgag gaatcttcaa atacagttgt aagatttctt ataagttgta 60 ttttttaaga ttaaacatat gtactagctc atgtctaatg catcataatt tgtagaggct 120 ttaaatgatt gactgatcct taaactttta tgtctcttct tccggctatt tcttttccat 180 240 actgccttaa ctttgttaac taacattgac tttttcatat gtgcaaatgc tggctagttt 300 ggtttcacaa tatctcatct tattgttagt tcgtcctcca ctgtattgtg tcataaattg 360 ttatctcaat tactgtcagg acattgatga aagagaagtt gagtaatggc gagacatctc 420 atgaacaacc tcatggcagt gttgcttggg aaggagatgt gtattctcaa gtgttgggaa 480 atgaaaaaag tggtaatgtc cgtggtttag gacttggtcc aaccccttct ctattatggg 540 gcggtaaatc ttccttacaa aatattaccg atgatggttt atctaatgag gctgcacata 600 agttagaaca agagataaag gagttaaagg acttgaacaa aaaacaggat gaaaatag 660 ctttgaatgaa aaaaaatcaa gatatgctag tttcagaatt aacatggatg aggcaagtca 720 tgtggaaata tgttcccacc aaattatgtg gccctcaaaa ctatggaagc actactagac 780 aggttattca atttcaaagt tttaaacttt tcttcttaaa attaagcttt ataagaaaat 840 tatgtgatct aatatgctt gatatatact aggttcctga tgccaatagt ggcaatgagc 900 911 dispatcher <210> 18 <211> 5963 <212> DNA <213> Nicotiana tabacum <400> 18 atggaggtac cggtgctagc tcggtgtacg aatactccga cgacgtcgtt tctaggatgt 60 aaagtgagtt tatttgattt tccgattaga agaaagctaa ataagaggaa ttataaggcg 120 aagttttcag tgttaagagt taaagctatg gcggagagga cgagtactga ggcatcagcg 180 gatgctagag agagagaaag tggagggtac acgggaacta cgatggaggt gacaacattt 240 aatcagagct ttagtgatgc gcaattgcca gtttgggaaa agattggtgc tgtcgtcaga 300 ctcagttatg gaatcggtga gttctcaact cgttttgcgt ttacttaatt agtctgtttt 360 tttttttt gttttttcg gtttgaattc tggtttcctt tttcaacgct atttttggga 420 aagaagacaa ggaatatgca tattagttga atagattcaa tgaattcaga attttatttt 480 gtggctttg gtttcctatt ttttggtca acatttggta tctgaaccca ctaacctgac 540 taatttgat tcgcgcctta gaaacttac tgtgggagat ttaagtgttc tttacaaga 600 ggattcaaac tcgaaacctt tgattacgtg taaaagaatt ttatcattc caacatacca 660 ttcaagggcg gatgtatcgt ttagcttatg agcttatttg aacccaattt tgactcggac 720 catatatatg tgttaaaaat acacaaatac atagatttg aacatagtaa 780 attaaatgga tatgtgatag aatccaaa ctctgaacct atatgttca aatctttact 840 ttagttttg gtggactttt tggttgttat gattagtcta tattgtgtgc gatttaatct 900 tagtagttac agtgtgagca gtgtaatact gttctcggtt tgatttat agagcaacaa 960 ttgttaaaat ataagcttaa ttttggttct atgattagg taatcctgcc catttactc 1020 gatttaattt gctatttact gtagtttttt ttattagaac agagttattt tgttattttc 1080 cttccccaaa actatctcca taaaagcctt tctttttt cttaaatttc gtgtccagtc 1140 aaataggttc ataaatttg aaacagaag atattaat agcatagtat aaatggattt 1200 cgaaattatt atagtagtat atacttatag tttaactttt aagttttaat aataacaagt 1260 attcggtaga acaatggagg tgcaaaatgc atttccgtat ttattcattt gcatgtaaag 1320 actagagtta gatgaaatgg gaatgtgtaa cgagaactta gtgctagaaa gaatagtaat 1380 taatgagaga aagtgaattt acaagtgatt cttggagttg caagtaaagg gaaaggaaaa 1440 gctaaatttg ctttctgtat atgtcattaa agtgcttcta aaaatggaaa agctaaggtt 1500 tttcttcttt ctaactttta ggttttatag ttggtaggac aaaaatgcc aaaaataaaa 1560 aaacactgca atatttttgc agtcaattta atcatgtttc caatcacatg ttaagtggta 1620 ctttgtttcc taagtaagaa atataagcaa ttgtcaggaa taggattgta tttatataaa 1680 caattgtaga taaaattgaa tactttttatt gttttgctc atacttggct attgaggaaa 1740 acagaattgt agttaggcaa tttgtcagaa tgcattagaa gaagtgtaga tgcattttag 1800 aatgacagaa aatacaaatt tatttacatt tttaagaata gtataaaata agtgatttta 1860 taaatattta gaacatgata atagaagaaa ataggcgaa attaaaaaa gaataaattt 1920 ggagccgaaa ttgatcattc taatgcttgt gtgctaacgt tgaatagctt taaatttagc 1980 aacttgttgg taatttttta aatttgtaga aatattttgt tttttgtttt ttctttttta 2040 ataaaccaat tttatttgtt aacgcaacta catgcttcta tagaattaca tttggaaaag 2100 ctttttagcc caaatactgc caaaaataat taaatgctaa aaacaaaata aacaaataat 2160 aactccatcc cgtgccactt aattagaagt agaattgtac ttaccggagt aatttgaact 2220 ttggcaatag ttgttaaaat atgttctagc ctaactagct gtactaaggt ctcaaaaact 2280 ttgaaatctt gaatctaatc ttcctcaaag aagaatagaa tttaatcatt gtactcgagt 2340 aaattgaaca ttgcaattat tatcatagta catttcccca aaatgtagta tgtccattgt 2400 aatactacat attcattttt tttatactt ttttaatacg gtgttatctt ccactaatat 2460 atatcgggta actctgacac catacataaa tatgacctag tgttttaat tccggtgtat 2520 ttattgttt gttgcatgta tttccatccc attaaatttt ttggatatct ttacagaaga 2580 aaaagaaatt aaagtccttc aatcattctc tgatgttcag tttgtttttt gcactcaggc 2640 atatatggag caatggcttt agcaggaaag ttcatatgct caatgacagg aattgactgc 2700 acaggagggt tcagtccatc attagatgcc attgttgaag gactaggata tgcagctcca 2760 ccaattatgg ctcttctatt tatactagat gtacgttcaa gattccctgt ttttctttat 2820 gcacataaca attaagtgtc ccaaatcagc cgacgaaatg agttgttatc ttcttatata 2880 gtcttagata attctcacat catgagattg tgtcggagcc aaactcctct cttggtttac 2940 acaatgttgg gctcctatgt tttgttgtac acgcaccata tgtccaatct tgggcgggcg 3000 agggtgttaa gctagttgag gaaatgaact gttatctttt tatatgatct tggacgattt 3060 tcatctcata agctaacttt tgggactgaa ttaggttcga ggttcttttt cgttacaata 3120 tttaactgca ctcttggctt cttcaacttg aattaaattc ttctatctac atatggcttt 3180 actaactctg aagtacaaaa atgcaagaat attagcctta tgtattgcag ctatactacc 3240 tttgtccata tgacattctt ggatcatata atggatgttt tataataatt tgcggtgttt 3300 ataggatgaa gttgtgaagc tgtcgcctca tgctcgagct atcagagatg tagaggatga 3360 agagctacgg aatttctttt atggaatgtc accttggcag gtaaaaattt ctcaatatga 3420 gcatcaataa ttatgttaca atacagggaa gattgatact taacggattt tgatgcaatg 3480 gtgtagttca ttctgattgt ggctgctagc tctgttggag aagagctttt ctaccgcgct 3540 gctgtccagg taagatgtat atcatccatt tatggtatac aatcgcgaat tcatttgtac 3600 atttgcagcc ttgtgttttt tccatattgt ttttcctatt tataatggta acaacagata 3660 cctggaaatc cttttggtga attttttttt ttgtgcttca gttttccaat gcatatatttt 3720 gcttattaaa agaaattaat atagaaagaa aaaagaatat tagaaaaaca tggaaaatag 3780 aaagaggctt aaggcgtgta gaacatagtt atctaaaagt aattgctgtt gttttttgtc 3840 tgatttaatt cacttctgaa gagccaacaa cagccaacga cgttcttatt catacaacag 3900 aatctcagaa agtgtgaaat cttttcagaa tagacatgtc ttttgttgat aaccactttc 3960 atattacaaa ctatccaaca atattatcaa cagcgtaatt atttttaact tccataaaaa 4020 atgcatgtca aaaaggttat ttttctctaa gttatgtgat tctttcctcc tctctgatta 4080 tctttgtaat tacagggagc tttagctgac attttcttaa ggggcagtgg ttttgtgact 4140 gatgctagag gaatggcatc attggtacgt cggattctat ttctaaacat ccttctccaa 4200 gttacattat ccaaaaattt tccattgctc tgacacattc tgtttcctca aaatgtagac 4260 tggtgttttg ccaccgtatg tcccatttgc tcaagcgttt gcagctgtaa ttacggcagc 4320 tctcacgggt tctctatatt atatggctgc ctctccaaaa ggtaagtttc acaggctgca 4380 gtttatatta tatatctgcc tttcattgtc ctctactcta acatgcttaa tcacttagct 4440 atttgttgtt tcacttttca gatcctacct atgttgttgc accagtgctg aagtcgcatt 4500 caggtcgtga agatcttaaa aaactatttg caggtttgac atctcttcta tgctcatgac 4560 ataagcaaga tagctctcac ttggtgatca tttgagtcta aacttaatta acataatgat 4620 atgaatctgg ggatttgggg tccaactaat aatgctaatg ggatatgaag aattagtatg 4680 gctgattgca actaagaata gaagagctga agcacgttcc ttagcctagt ctacatccaa 4740 agttgacaga cttaaaatac aacagtctcg acctactttg aatcaaaaga aaggcgaaag 4800 gtttggcaac aagcgaatcg cttttcatca tagttgcaag gattccaaac ctgctccctg 4860 gtcgttgctc cactgcatct ggtcatctct agtcaccttt ggaaggcagg aagtgtgaca 4920 atatgcatgt tttctttact ccatcgggcc ttctcaggcc tttccttgta ggagtagctt 4980 cttaaactat tttaaataat atgagaccac caaggcaggc actcgttctg tcaaaagcaa 5040 cggacttact tctggttcta ggtgttagcg tgtacgaaag attccccggc tctctgtata 5100 cttaaggtgg tctcttcaca agtgtcacca gcagtggcag agccaggatt ttcaccaaga 5160 ggattcaaaa tataaagagg tgaacacttg gagaagccaa ggggactcaa cctctactat 5220 atatacatac aaatattttt ggtcttgtat acatagtgta attttccgtc gaagggagtt 5280 cggatgaacc cccttccacc accctagttc tgcccacggt caccagagag agcgagcaca 5340 gattggatta aagaaaaatt ggtttctact gagcttatcg aaccaactca tatcgttaag 5400 atatgataag ttttatatta tttgatgtaa ccacctgtat cttatctcca tttcaaacct 5460 aacaaatcct actccctcta cataaacctg tacactacaa aatatcagaa gcaagtaaac 5520 aaagcatttc tgctctccaa cgtgtgatcc tttagttgaa acagataagt gcatgatatg 5580 ttaattattt catctgcctg aatgttttgc agcttggtac gagaggcgac agatgaagaa 5640 gatatactct cctttactag aagccatgtt agccctttac cttggttttg aatggatcca 5700 ggtaacatga atttcacatt gtatttatt ctgatactta aaaggttatg tctacatgaa 5760 attttggtca ctaaagctaa tttgaacttc tacccttgct atgcacag acaaacaa 5820 ttttgcacc gataatcaca catgggat actctgctgt tattctggga catggacttt 5880 ggaaaatcca cgatcatcgg agaagactac atcaagaat ccaaactt aaaacaagaag 5940 gtaacaattc aagaaacttg this 5963 <210> 19 <211> 2193 <212> DNA <213> Nicotiana tabacum <400> 19 atggtaaatt tcaaaccc ccaaaactcc ttatattctt tttaaaaa atttcatca 60 atttgattgc ttcactct ctttttgttc attatttta aaattgtgtc tctttttgg 120 tattagggag ctggaatacc tgatgagag gaaatatt ggccactatg gttaaagcca 180 ttgcttaaag aaaaattctt tggccattgc aaattacatg ctgattctca caagagtgaa 240 tgcaatatgt attgtcttga ttgtataaat ggccctcttt gttctctttg tttagcacat 300 caaaggacc atattgctat tcaggtatct catttttcct tcatagaaaa atggtctttt 360 tatgtcaatt tgtaacaaat ttttccttta gttcttcctg tttagcagca actaatgctt 420 gtattagggt agtctgttta tgtcacatca catccctagg ggtgcggcac tttttcggat 480 tccgagtgaa tatgaaatct cttgtgcatc ggcaggcctt ttggtataca gtgtatacaa 540 tggcgaagtt agaaatttta acgagggtct atagctacac attctatatg gtgacgaaag 600 ccagaaattc taacaacggg gttcaagaaa atgctaaagt gtcacaccta aagtttgatc 660 atgttatttt aagcaatttt taacctgcct ttgccactat actaaaaatct ttttttatgt 720 aaaaaatttt caaaatgcaa aaaagtttat ttttgaccta cttttgagta taatttttcg 780 atgaaagtga ttcaattgaa cccctaaatt gtcattttac atggtgtata gctttgatat 840 gccaaagaag aagtcttgaa attatgaatg attgattttg agtgatctgt ttttgtttgg 900 ttggacagat aaggaggtca tcataccatg atgtgataag ggtgaatgaa attcaaaagt 960 atttggacat ttcttcagtc caaacataca ttatcaacag tgctaaggtt gtctttttga 1020 atgaaaggcc acaacctagg ccaggcaaag gtgtaacaaa tacttgtcaa gtttgtgaaa 1080 ggagccttct tgattccttc aaattctgct ctcttggttg caaggtacaa tactgtttac 1140 tttttcaagt ctgatatata ttaattatac acggttataa acatattata tagattatgc 1200 atatgttata catacggagg ctatttttaa tttaaacagt tgagtggatt gctatttaag 1260 ttaattgttc taaaagaaaa ttcccttcta gagtttcatt aaattgttcc atttttttca 1320 tcttgaccaa attttcttgc attaattttg acttttactg gcattgaatt gaattaggcg 1380 tgtccaaata tattgaattg cgtatatttc aaggtatatt ttggtttaat ggtcgttgat 1440 agtgtggtt gacaaatgtg aagcaattaa atggggcgtg gaaattgcac tcatatttac 1500 tagtctattg ataggaaaac tttggcataa tcaactttt ttaattggaa atttcaaagt 1560 tgttagacat tgtaattatc aatgactctt tcttgcagtt attgtgtacc ccaaatggaa 1620 atgatattca aatctttgat tattagcaat ctttgatata caataaatat tatgtgaatt 1680 attgaattgg tccgaataga gcagaattct atgttttgtg ttgatggtat atattgtctt 1740 ttcgtcttt ttcgttttct tgagccgagg gtctattgga aacctcacct ctactctatc 1800 ggggtagggg taaggtccgt gcacacacta ccctccctag actccttacc tgcgggattt 1860 tactgggttg ttatccgaac agaacagaat gaatataggt gatttatata gtcgattcta 1920 ccttgatcat tgcaggttgt cgggtcctca aagaacttcg ttaagaaacc gaagcaatta 1980 tccgcgaaaa ggcggcggtc gatggtggcg gcatcggact ccgatgactc ttacagcagc 2040 agcagccatg gtaggtacaa gagccacagc aacaaggtcc aaagttttac tccgtcgacg 2100 ccccctccaa cttcagttaa ttacaaaacg gccaagcgaa gaaagggaat tccacataga 2160 tccccaatgg gaggactact catagaatat taa 2193 <210> 20 <211> 8752 <212> DNA <213> Nicotiana tabacum <400> 20 atgtcttccg ataacttcac cgacaaaaac gccgtcttcc gcaagctcaa agccaagccg 60 gacaacaagg tttgcatctt aattcatcga attttgtttt tgatttgatc gaaaaattat 120 gttttttgaa tttttatcta ttttctcgcg tgttttttcg tttgaatcga ttgaaaattg 180 cgagatctga tttagatcta gtgtttgtgc tgagtagatt gtagtttctg agcgatctaa 240 gtcagctttt accttttttt tttttttgaa ttttatcaga tgtgttttga ttgcaatgcg 300 aagaatccta cgtgggcgtc ggttacgtat gggatcttct tgtgtattga ttgttcggcg 360 acgcatcgta gccttggtgt tcacatcagt tttgttaggt acgatttaaa gtttgatttt 420 ttactcgtct tcgtttttgg attcatgagc tgatttttag cttgataatt tgttttcatg 480 atgagtggat tttgttcgga gttctataag gcatggtttt tggttttata aatatatcga 540 aaaaatgatt agaaatgttg aaatcaatag aatcaacagg ttcgaagccg tgacttctgc 600 cttataaata agtggctgat ttaaaatgtg gactgaggta agttcggacc cgctgtcttg 660 agtaggggca gatgtactgt tcggggtacg ggtttggtag aactcagaag ttttggccta 720 agccatgtat gtttcctaag aatttcactt aatatgaata gattattaat ttagaaccca 780 ttaactcata attgcctaaa ccccgcacct ttaaacttca aatcatggat ccgcctatgg 840 tcttgaactc aagttatatg ttgttgaaat ttatttaaaa atatgtagtt ataataggag 900 tagtggggtg caatggtatt ctacttgatt ctaaatcctg aattcgtctc tgtttattgg 960 acgctgaaat tttaattttt tgctgcgttc cttacgaggt gctttaggca atggtctttg 1020 atacaaggga acagattcta tactgtgaaa agaattatgc taatgatgaa agcattttaa 1080 agatctaatt atgtggcatg gtcccgggcc ctaaactcct cttaattgta gaatatgtgg 1140 acgttgagcg cgccattggg ctagatgcag tgaaatccat tggttctca actatcttgc 1200 tttgttttct ctccttgata tgtggtttt agggttacat gtggtccaag ttttggtgg 1260 tagttgatag atatgtattg ctcctattgc tcaatattgc aattacagtc agacctctct 1320 ataacatccc tatataacaa cacttcacta taaaagccaa gctttccgg aatgttatgt 1380 tataatatat gttctatata acaacactta gttataacat ccaaaaatat tcggaaaaa 1440 cgaggctgtt atagagaggt ttgactcctg agcacgtatc aaaagattgc atctttttgat 1500 gaatatgtgt ccgacaatct tgattatgga ttaagcttaa tcttatgctc cgtattcgat 1560 cattgatgca aacgataaat tttgaacaag aaatggtcaa caccacatga tttcctttta 1620 tggcggatat tggtgcgcat accccttttc gagaaagact attgatatat gacctcttcg 1680 aagctacaac cagtatgcga atatggacga tctagtatga atcccatatg tgtttcttat 1740 acaataccat tccgcagtt tctggttcga aaccattatc ttcatcccat atgtgccaca 1800 acagcttcat tttgcccaac aattggtatg accaccttaa gagactttat gtgtttgcac 1860 caagggcttt ggtttagcgg taagagcgca atgtttgatg tgtggattag gcgcatgcca 1920 caggttttga atcctgtagc ggaaaaaagt ttggtgttta agtggagaag ggtagaaggt 1980 ggccccttta tctaccgagt tccgaaccgt gcaccattgg cacttgtgaa ttttttgatt 2040 attagagaaa tggagagatt tttcttgtgt tacttgcagt gtcctaccta attacctatc 2100 atggttaaca aaaccgacaa agtgagccat cataatctgg ctacttttat tcttaacaca 2160 ttttcttcca tctcattgaa aaatccatat tctttctttg gaggagggtg tgaatgaagc 2220 cttctcttt catccactac tagttcttca cattttttcc gatgacggtg gtgtcggggc 2280 cagtttgaat gctccttaat tattctaccg ggtacctggt tacctcccac tagcacatgt 2340 accaggtgga ctctgtctac ctaggcttgg gcgtatgtga agaaatcacc tattggtttt 2400 ttggcttcac taggattga atatgagact tcatgattct cttcctactt cattgacccc 2460 taggtcacac ccttgggtgc atgctagttc ttcatattat ataatgtcac tgctattttt 2520 ttcaaatgac actgccaatg ggatgaatct aagagaggaa gaacagaaaa gtaagccgag 2580 gaagaacgga aaaggacgag gaagaataga aaaggagaag ttctaaatgg ccatgtcaga 2640 cttcttggct gacatgcgtt ctttttataa caaccggcat gaccactgca tcacactttg 2700 ggcctcgtca gttccattgt tgctcatctc acctcaccat ggtagctgga cttcctacc 2760 tctaacaatc agcatgtcag tatgttagtc aagcgaagac aataatagca ttttattacc 2820 tttgaaaaa atgatgacaa caatggcatg gttagtgatt aatctataca ttgttaaacc 2880 taacctgatc atttgattga ttatgagaac aattggccat cggagattat ggaaggccat 2940 gaaggctaga ttttgaggta tttttgtggg tcggcttgct tcaaaagaga aaagagggtg 3000 ggtgggggtt gggggtgggg tttgaaaggc agaagtggaa tgtagagttt gaatgcatgt 3060 atctgttagt atgtgttaga gatagagagg ggtgataata ttgctttgca tttcctttat 3120 catggttgag atgttcatta tgggccgaga gttatcttct gacccacccc cttttcgttt 3180 gtaatgtttt gaggtgggaa tgggtatggc caaccggaaa caaataaatt ctgcattttc 3240 tttgttattt tggcattact aactgtcagt tctctgatga attgattctt gcagatcgac 3300 aaatttagat tcatggtctc cagagcagtt aaagatgatg tactttggtg gaaacaaccg 3360 tgctcaagtt tttttcaagc agcactgatg gacggatggc ggcaaggttg aagccaagta 3420 tacctccagg gctgctgaat tgtataaaca attactatca aaagaagttg ctaaaagtaa 3480 agcagaggat gcaggtttgc cagcatcgcc ttttgcctct caggctgtgc agagtacgaa 3540 tggattttct gatgttaaga ctagtgaagc tccaaaagaa acctcatcat ttaaggaaga 3600 aactcctgct tcgcccaag catcacaatc agttgttact acttcatta ggaaacctat 3660 aggtgcaaag aaatctggga agccaggtgg tggacttgga gctcggaac ttactaaaaa 3720 gtcttctta gtcttatgag ctagagaaat gattgcagggg tataaattgc atattctgaa 3780 atgaagactt atatgtgcgg tttagagtaa gctgtttaac aaatttgctc caccaatttg 3840 gatttatta attgattaag catctttac cggtgtacag ccaagtgaaa gtctctacga 3900 ccagaagcct gagaaccgc ctgttcaagt ttctcctcc aattctacaa gtaatgcatc 3960 aactgttggt tcatcattcg catctcgctt tgagtacaca gatagtcc aacccgctga 4020 gatgagttct ggaggccctc gtgttcttaa ccatgtatcc cctccaatgt cctccagctt 4080 ttttcggac tacggaatgg acagcggtttt cacaagaag acagttcaa attcgtcaa 4140 agttcaagtg agtttgtatt ctagttttgt ttcaatcat atgaggattc taatgaactg 4200 ttacagttga acttaaatgt tgacatgatt atatgtgaga tatgaagaa gtggtgtcgg 4260 acttagttac aaggtaaga tataagcttc atttttcacc ttatatcac caggaatatt 4320 agttccttgg tacgactata cctttttcac gtcctattac ctagtataca catttgatct 4380 atctgaagca ggcttgagtt tttctgaacc tgcataagtg ctatatccaa gatgagcaat 4440 ttaatgtata caattaaaaa aagggcagca tgttgcacta agctcctgct atgcttgggt 4500 caggaaagga ccggaccaca ttgggtctga tgtacgcagt cgccttacct tgcatttata 4560 caaaaggctg tttccatggt ttgaacccat gaggtcatgg cagcaacatt tattgttgcg 4620 cggaggtttc ccttccattg agcttctgaa agattggggg tagtgatttg tgaaagattt 4680 actgctttat gatcctattc cagcatcctg cactgtgaat aattacaagg attgtcactg 4740 aattgaaaaa aaattgtttc ccttggatct ttggatgctt ctcgacggtc tatttgcttt 4800 tctttttttc tttttaaatg aaactaccca ttaccactct tgaaatactt attgccccac 4860 tcccttcccc actgaagaaa cattttcatt aggttaaccc aaatcgctag tgtctctacc 4920 tttttagtca ttcaagaaaa agtaaatgtt catcctaaac attaccagtg caagtttcaa 4980 gtgtctgctt actaatgaaa aagaaaaaga aacaactttt ggatgttttc tttttgaaat 5040 ataggttgcc tggaatattc tttgttgttt ttaaacctca tcttgtgctg gatttgtcct 5100 ttaatttgtg atttactgct ctcactcagt tcttctttg ctcctgatag acgagttgac 5160 tgctttgcct aagttatttc tgtagtcttc aagttttcttt ttctctcttc tcctttatca 5220 tcttcactgg ccgtgttat ttcactgtaa ctcacatcct atttaatca gattgaggaa 5280 actgatgaag caaggaagaa gttttctaat gcaaaagcca tttcatctgc ccaattcttt 5340 ggtgataaga gcaaagctga aatggaagcc tcagtttctc tgcagaagtt ctcggtatgc 5400 ttgcttattg tagttaacat gcccatattt ttaggggcct tgctggtttt tcagtcagca 5460 tgcaaacaat gaaaaaccct ctcccttccc tcattcatta tgatagttag ggtcttgtcc 5520 tactaattag aaataactat tatcgcaaca acattatgcc ccatgttctc cttttttctt 5580 cccattccct tttgtccttt aatgcactgt acttcttccc atgctatcat tttgctgggt 5640 tttttttt tgatgaagta agagaatttc attaaaggca tcaagaagat gcatagcaaa 5700 agagagtaca agtaataga gtgtgtctgc tcataaacat aacaaaaaga aacctatgtt 5760 aaaactaagg agcagacaca gtccaaaaaa tgatcacaac tagatacagg tgcctgatta 5820 agccaagtaa aaaggttaaa taaacattta gctttaagag catgatttgg atttgatatc 5880 ccatcaaaac atctgctatt cctttcagtc catatacacc agaaataga agcggggacc 5940 aaaagtcaag ttttttgat ggatttgcca actctccaag agcaccagct ttcgtaggcc 6000 tctttaatac tattaggcat gacccaggct agtccaaaaa ttgagaagaa catactccac 6060 aaatctgctg caactgcaca atggagaaa agatgcctca cactctgc cttcatctga 6120 cacgtagcac ctattcacaa tgtagatgtt cctccgactt agattatctt gagtgagaca 6180 tgcttcatat aaggttatcc aagtgaagcc aattacttta ggtggaagtt tatagttctc 6240 cagatgagtt tccatggcca gttgtcaatc tactccttgt ttgagcacaa atgtacataa 6300 cctttcttga cagtgaatat cccatcatgt tggccattcc ataatagtct gtctctcctg 6360 aggtttgata gtgacccctg ctagcttaga ttgaagctcc agatagtcat ttagctccca 6420 gtcctgtaag tttctccctg tctgtaggtc ccatgagttg ttgtgccata ttgacccac 6480 tgtggagttt tgattgctag ctatgagata taaatttggg tagtcttctt tcagagtctg 6540 gttccctaac cacttatcct tctagaatga tacatgcgct ccattcccta actttattga 6600 acaattgagc tggaattcac cccataggct tgagatatgc ttccagggac cagtgccatg 6660 tggtaatctg ctctgtttgg tgcaccattg actcagcacc ccatactttg ccatgatcac 6720 atctttccac atcccaatgt tctccatgtt gtacctccaa tgccacttaa ttagcatgct 6780 cttgttgtgc aattttaagt cttttatgcc caaccctcca tgctcctttg gcaaaatcac 6840 tcttggccat ttcaccagat gaaatttgtt gttttgacta tttcctcccc acagaaagtt 6900 tctattaatt ttgtctaacc ttttctgcac cctggctggt ataggaaata gggacatgaa 6960 gtatgttggt atgctgtcca ggacactgtt aatgggtgtt gttctgcccc caagtgaaag 7020 atactgcatt tgccaagttt caagctttgt ttcaaatttc tctactatcc cattccagat 7080 atctggtgac ttgactttgg ctccaagtgg tagacccaaa tatgttgtgg aaaaagatcc 7140 cacattgcaa cccattagct ctgctagctc ctccatattt ggtaaatagt gctcttaaat 7200 ctgttcatgt ggaggccgga tatagcttca aaaatcatga gtgtgaggtt aagatagagc 7260 acctgcgacc tatcagctcc acagaaaata agtgtgtcat atgcatacaa gagatgggag 7320 acactaattc agctcccaac agaactttcc accttgaaac cctccaacca gtgtatttgg 7380 tttgctttgt caagcatttc gctaaggcct tacattgcca aaatgaacaa aaaggatgag 7440 aggattccgt tgacaagaac tgaatacttc acagtggtta cacaaaactt gatccacctg 7500 atccaacctg caccaaaccc catcttccta attatattga ataggtaaga ccagtttagc 7560 tggtcaaagg ccttttcaat agcaagtttg aaaaggagac ctggctgccc tgatttctgc 7620 ctccagtcta gtacctcatt agcaattagt gcagcatcag tgatttgtct tgttttgatg 7680 aatgcatttt gatgccctga gacaagcttc ccaatcactc ttttcagtct ttctgccagg 7740 attttggagg caatcttgta acttaaaaag agactgttca acagttactg tgagagcctt 7800 ctacaatgga acttattaat taccagtact tgggttgcag aaccacattt atctgtatgt 7860 tgaagatacc ttatcgatgc tgtttactga ggcgtagatt gaaggatata tacgatggat 7920 aatttttgaa attccattca tggataat ggtgaagacc atttgccata acttgagata 7980 atggtgtcta gctgacctct tgtttgttcg cttcataatt gcttatgttc atttgcataa 8040 caagttatct actggtccga cgtactgtgg atagtttac ccctcaggtg ttttatac 8100 ttaatctcat tttgaggag cttgtaatag ctacttcagc aaatgaatat gatttgtaag 8160 ctaaaatgat ccgcacagct gatagcatta gtcctaatct gatttattac ctgttacatt 8220 tgcctgtcgg gttgcttccg ctagtgctta ttgtatattt acttcgtcaa aaaaaacttt 8280 gtgcttattt tgtattttgt ctgacccatg tctgaagaat cctttagtgt caatttctaa 8340 acgagtgcaa ctatattact tgagcttgac ctgaaaagcg ataagttcac ctagaaatgt 8400 atgagtccaa ctcgtgctcc taaaaatgtg ttctctac tattcctcag ggttcaagtg 8460 ccattcaag tgcagacctt ttgtaacg atgatagagc agatttggac ctcactgctg 8520 gtgatctcat taaccggctc tctttccagg tatagaagca ataatacac aaatgtaaac 8580 gcttttcaat tcccataagc aaattttgaa cggattgctc ttcgtttgt ctctatgttt 8640 tcaggcacag caggacatct cctctctgaa aaatattgct ggagaaactg gaaagaaact 8700 tggctccttg gcagcaacct taatgtccga ctttcaagac agaatcctgt ga 8752 <210> 21 <211> 471 <212> DNA <213> Nicotiana tabacum <400> 21 atggcatctc tctcatggtg gaatcctgct cctgccacga ctgcaatggc agcttgttct 60 ccaactccaa catcctgtaa aacctctaac tcattagcac tgccgcgctc tgtgtttgtc 120 agcaagcaag caaagttaat gaaacaagcc aatggtcttt tggttataac acagcaacag 180 tcaaagaaga agaatcattc attcaccaat tccagaagga atacagcat tcagtgtctc 240 tcacaggaac agaaatggac tcatgaaggt tccattaccg aatcgctccc caatggcatg 300 tttagggtca aattggataa tgcagatgtc gttctgggat acatttctgg gaagatacga 360 aagaatttca tacggttgtt gccaggcgac agagtcaaaa ttgaagtaag tcggtatgat 420 tccactaaag gacgcatcat ttatcgtctc cgcggtggcc gagaaggcta g 471 <210> 22 <211> 768 <212> DNA <213> Nicotiana tabacum <400> 22 atgcaaacca gagttggtag tagattggtc actgaacagt atcacgatga agaagtacgg 60 ccatatattc aacattaat gatgctcag aattgctc cagcccaaac ttatgatat 120 caatctaata tttgacaa ttctgttggt actggagctg agcagaacaa tgaatcaggt 180 ccttcagaga atagtcgtaa tcaagctatt ggaaaaaagg gtcgaaagct tgctagcttt 240 ctaagtatca ttgctagac cccagagctg acaccgttaa ataatga tggcgagta 300 tttgacaag aagaaaagaa gaaattggtg gagtttgtga ggaaaaagtt ctcgattcca 360 gtatgtagag aagagtttat aaaaagtca atagggaaa atggaagga ctataaatgt 420 gatttgaga ctatgtatgt gaccaagtat agagcaag atgccttgat gaaaataga 480 ccaagtcaca taccaaggga tcaatggact gtctcgtct tgtattggct ttctgataaa 540 gcaagaagc gcagtcaagc aaatagaatc agtagggcta aaaaagat gcctcacaca 600 ggaggatcca aaagcatagc aaccttgatg atgaaagg ctatagatgg atagagcct 660 acacgtgctc aagtttacat attackcat aaagcgta aggatggtag accattggat 720 gaggaatctt caatacagt tgacattgat gaagagag tgaggta 768 <210> 23 <211> 480 <212> DNA <213> Nicotiana tabacum <400> 23 atggtagacc attggatgag gatcttca atacagttga cattgatgaa agagaagttg 60 agtaatggcg agacatctca tgaacaacct catggcagtg tgcttgg aggagatgtg 120 tattctcaag tgttgggaaa tgaaaaaagt ggtaatgtcc gtggtttagg acttggtcca 180 accccttctc tattatgggg cggtaaatct tccttacaa atttaccga tgatggttta 240 tctaatgagg ctgcacataa gttagaacaa gagaataagg agttaagga cttgaacaa 300 aaacaggatg aaaatagc tttgatgaaaaaatcaag atatgctagt ttcagaatta 360 acatggatga ggcaagtcat gtggaatat gttcccacca aattatgtgg ccctcaaac 420 tatggaagca ctactagaca gttcctgat gccaatagtg gcaatgagca agcaaccta 480 <210> 24 <211> 1113 <212> DNA <213> Nicotiana tobacco <400> 24 atggaggtac cggtgctagc tcggtgtacg aatactccga cgacgtcgtt tctaggatgt aaagtgagtt tatttgattt tccgattaga agaaagctaa ataagagga ttataaggcg aagttttcag tgttaagagt taaagctatg gcggagagga cgagtactga ggcatcagcg 240. gatgctagag agagagaag tggagggtac acgggaacta cgatggaggt gacaacattt aatcagagct ttagtgatgc gcaattgcca gtttgggaaa agattggtgc tgtcgtcaga ctcagttatg gaatcggcat attggagca atggctttag caggaaagtt catatgctca atgacagga ttgactgcac aggagggttc agtccatcat taggccat tgttgaagga ctaggatatg cagctccacc aattatggct cttctattta tactagatga tgaagttgtg aagctgtcgc ctcatgctcg agctatcaga gatgtagagg atgaagagct acggaattc ttttatgga tgtcaccttg gcagttcatt ctgattgtgg ctgctagctc tgttggaga gagcttttct accgcgctgc tgtccaggga gctttagctg acattttctt aaggggcagt 660 ggttttgtga ctgatgctag aggaatggca tcattgactg gtgtttgcc accgtatgtc 720 ccatttgctc aagcgtttgc agctgtaatt acggcagctc tcacggttc tctatattat 780 atggctgcct ctccaaaaga tcctacctat gttgttgcac cagtgctgaa gtcgcattca 840 ggtcgtgaag atcttaaaaa actatttgca gcttggtacg agaggcgaca gatgagaag 900 atatactctc ctttactga agccatgtta gcctttacc tggttttga atggatccag 960 aaaaaaca ttttgcacc gataatcaca catggtat actctgctgt tattctggga 1020 catggacttt ggaaaatcca cgatcatcgg agagactac atcaagaat ccacaactt 1080 aaacagaag gtaacaattc aaaaacttg taa 1113 <210> 25 <211> 675 <212> DNA <213> Nicotiana tabacum <400> 25 atgggagctg gatacctga tgaagaggaa aaatttggc cactatggtt aaagccattg 60 cttaaagaaa aattctttgg ccattgcaaa ttacatgctg attctcacaa gagtgaatgc 120 atatgtatt gtcttgattg tataaatggc cctcttgtt ctctttgttt agcacatcac 180 aaggaccata ttgctattca gataaggagg tcatcatacc atgatgtgat aagggtgaat 240 gaaattcaaa agtatttgga catttcttca gtccaaacat acattatcaa cagtgctaag 300 gttgtctttt tgaatgaaag gccacaacct aggccaggca aaggtgtaac aataacttgt 360 caagtttgtg aaaggagcct tcttgattcc ttcaaattct gctctcttgg ttgcaaggtt 420 gtcgggtcct caaagaactt cgttaagaaa ccgaagcaat tatccgcgaa aaggcggcgg 480 tcgatggtgg cggcatcgga ctccgatgac tcttacagca gcagcagcca tggtaggtac 540 aagagccaca gcaacaaggt ccaaagtttt actccgtcga cgccccctcc aacttcagtt 600 attacaaaa cggccaagcg aagaaaggga attccacata gatccccaat gggaggacta 660 ctcatagaat attaa 675 <210> 26 <211> 1359 <212> DNA <213> Nicotiana tabacum <400> 26 atgtcttccg ataacttcac cgacaaaaac gccgtcttcc gcaagctcaa agccaagccg 60 gacaacaaga tgtgttttga ttgcaatgcg aagaatccta cgtgggcgtc ggttacgtat 120 gggatcttct tgtgtattga ttgttcggcg acgcatcgta gccttggtgt tcacatcagt 180 tttgttaggg gcagatgtac tgttcggggt acgggtttgg tagaactcag aagttttggc 240 ctaagccatt ttctggttcg aaaccattat cttcatccca tatgtgccac aacagcttca 300 ttttgcccaa caattggtat gaccacctta agagacttta tgtgtttgca ccaagggctt 360 tggtttagcg gtaagagcgc aatgtttgat gtgtggatta ggcgcatgcc acagatcgac 420 aaatttagat tcatggtctc cagagcagtt aaagatgatg tactttggtg gaaacaaccg 480 tgctcaagtt tttttcaagc agcactgatg gacggatggc ggcaagagga tgcaggtttg 540 ccagcatcgc cttttgcctc tcaggctgtg cagagtacga atggattttc tgatgttaag 600 actagtgaag ctccaaaaga aacctcatca tttaaggaag aaactcctgc ttcgcccaaa 660 gcatcacaat cagttgttac tacttcaatt aggaaaccta taggtgcaaa gaaatctggg 720 aagccaggtg gtggacttgg agctcggaaa cttactaaaa agccaagtga aagtctctac 780 gaccagaagc ctgaagaacc gcctgttcaa gtttcttcct ccaattctac aagtaatgca 840 tcaactgttg gttcatcatt cgcatctcgc tttgagtaca cagacaatgt ccaacccgct 900 gagatgagtt ctggaggccc tcgtgttctt aaccatgtat cccctccaat gtcctccagc 960 tttttttcgg actacggaat ggacagcggt ttcacaaaga agacaagttc aaattcgtca 1020 aaagttcaaa ttgaggaaac tgatgaagca aggaagaagt tttctaatgc aaaagccatt 1080 tcatctgccc aattctttgg tgataagagc aaagctgaaa tggaagcctc agtttctctg 1140 cagaagttct cgggttcaag tgccatttca agtgcagacc tttttggtaa cgatgataga 1200 gcagatttgg acctcactgc tggtgatctc attaaccggc tctctttcca ggcacagcag 1260 gacatctcct ctctgaaaaa tattgctgga gaaactggaa agaaacttgg ctccttggca 1320 gcaaccttaa tgtccgactt tcaagacaga atcctgtga 1359 <210> 27 <211> 471 <212> DNA <213> Nicotiana tabacum <400> 27 atggcatctc tctcatggtg gaatcctgct cctgccacga ctgcaatggc agcttgttct 60 ccaactccaa catcctgtaa aacctctaac tcattagcac tgccgcgctc tgtgtttgtc 120 agcaagcaag acacttaat gaacaagcc aatggtcttt tggttataac acaccaacag 180 tcaagaaga agaatcattc attcaccaat tccagaagga ataccagcat tcagtgtctc 240 tcacaggaac agaatggac tcatgaaggt tccattaccg atcgctccc caatggcatg 300 tttaggtca aattggataa tgcagatgtc gttctgggat acatttctgg gaagatacga 360 aagaatttca tacggttgtt gccaggcgac agagtcaaa ttgaagtaag tcggtatgat 420 tccactaaag gacgcatcat ttacgctc cgcggtggcc gagaaggcta g 471 <210> 28 <211> 255 <212> PRT <213> Nicotiana tabacum <400> 28 Met Gln Thr Arg Val Gly Ser Arg Leu Val Thr Glu Gln Tyr His Asp 1 5 10 15 Glu Glu Val Arg Pro Tyr With Gln Gln Leu Met Asn Only Gln Asn Cys 20 25 30 Ser Pro Only Gln Thr Tyr Asp Asn Gln Ser Asn Leading Asn Ser 35 40 45 Val Gly Thr Gly Ala Glu Gln Asn Asn Glu Ser Gly Pro Ser Glu Asn 50 55 60 Ser Arg Asn Gln Ala Ile Gly Lys Lys Gly Arg Lys Leu Ala Ser Phe 65 70 75 80 Leu Ser Ile Ile Ala Arg Thr Pro Glu Leu Thr Pro Leu Asn Ile Asn 85 90 95 Asp Trp Arg Val Phe Asp Lys Glu Glu Lys Lys Lys Leu Val Glu Phe 100 105 110 Val Arg Lys Lys Phe Ser Ile Pro Val Cys Arg Glu Glu Phe Ile Lys 115 120 125 Lys Ser Ile Gly Lys Lys Trp Lys Asp Tyr Lys Cys Asp Leu Lys Thr 130 135 140 Met Tyr Val Thr Lys Tyr Lys Ser Lys Asp Ala Leu Met Lys Asn Arg 145 150 155 160 Pro Ser His Ile Pro Arg Asp Gln Trp Thr Gly Leu Val Leu Tyr Trp 165 170 175 Leu Ser Asp Lys Ala Lys Lys Arg Ser Gln Ala Asn Arg Ile Ser Arg 180 185 190 Ala Lys Gln Lys Met Pro His Thr Gly Gly Ser Lys Ser Ile Ala Thr 195 200 205 Leu Met Asn Glu Lys Ala Ile Asp Gly Ile Glu Pro Thr Arg Ala Gln 210 215 220 Val Tyr Ile Leu Thr His Thr Lys Arg Lys Asp Gly Arg Pro Leu Asp 225 230 235 240 Glu Glu Ser Ser Asn Thr Val Asp Ile Asp Glu Arg Glu Val Glu 245 250 255 <210> 29 <211> 159 <212> PRT <213> Nicotiana tabacum <400> 29 Met Val Asp His Trp Met Arg Asn Leu Gln Ile Gln Leu Thr Leu Met 1 5 10 15 Lys Glu Lys Leu Ser Asn Gly Glu Thr Ser His Glu Gln Pro His Gly 20 25 30 Ser Val Ala Trp Glu Gly Asp Val Tyr Ser Gln Val Leu Gly Asn Glu 35 40 45 Lys Ser Gly Asn Val Arg Gly Leu Gly Leu Gly Pro Thr Pro Ser Leu 50 55 60 Leu Trp Gly Gly Lys Ser Ser Leu Gln Asn Ile Thr Asp Asp Gly Leu 65 70 75 80 Ser Asn Glu Ala Ala His Lys Leu Glu Gln Glu Ile Lys Glu Leu Lys 85 90 95 Asp Leu Asn Lys Lys Gln Asp Glu Glu Ile Ala Leu Met Lys Lys Asn 100 105 110 Gln Asp Met Leu Val Ser Glu Leu Thr Trp Met Arg Gln Val Met Trp 115 120 125 Lys Tyr Val Pro Thr Lys Leu Cys Gly Pro Gln Asn Tyr Gly Ser Thr 130 135 140 Thr Arg Gln Val Pro Asp Ala Asn Ser Gly Asn Glu Gln Ala Thr 145 150 155 <210> 30 <211> 370 <212> PRT <213> Nicotiana tabacum <400> 30 Met Glu Val Pro Val Leu Ala Arg Cys Thr Asn Thr Pro Thr Thr Ser 1 5 10 15 Phe Leu Gly Cys Lys Val Ser Leu Phe Asp Phe Pro Ile Arg Arg Lys 20 25 30 Leu Asn Lys Arg Asn Tyr Lys Ala Lys Phe Ser Val Leu Arg Val Lys 35 40 45 Ala Met Ala Glu Arg Thr Ser Thr Glu Ala Ser Ala Asp Ala Arg Glu 50 55 60 Arg Glu Ser Gly Gly Tyr Thr Gly Thr Thr Met Glu Val Thr Thr Phe 65 70 75 80 Asn Gln Ser Phe Ser Asp Ala Gln Leu Pro Val Trp Glu Lys Ile Gly 85 90 95 Ala Val Val Arg Leu Ser Tyr Gly Ile Gly Ile Tyr Gly Ala Met Ala 100 105 110 Leu Ala Gly Lys Phe Ile Cys Ser Met Thr Gly Ile Asp Cys Thr Gly 115 120 125 Gly Phe Ser Pro Ser Leu Asp Ala Ile Val Glu Gly Leu Gly Tyr Ala 130 135 140 Ala Pro Pro Ile Met Ala Leu Leu Phe Ile Leu Asp Asp Glu Val Val 145 150 155 160 Lys Leu Ser Pro His Ala Arg Ala Ile Arg Asp Val Glu Asp Glu Glu 165 170 175 Leu Arg Asn Phe Phe Tyr Gly Met Ser Pro Trp Gln Phe Ile Leu Ile 180 185 190 Val Ala Ala Ser Ser Val Gly Glu Glu Leu Phe Tyr Arg Ala Ala Val 195 200 205 Gln Gly Ala Leu Ala Asp Ile Phe Leu Arg Gly Ser Gly Phe Val Thr 210 215 220 Asp Ala Arg Gly Met Ala Ser Leu Thr Gly Val Leu Pro Pro Tyr Val 225 230 235 240 Pro Phe Ala Gln Ala Phe Ala Ala Val Ile Thr Ala Ala Leu Thr Gly 245 250 255 Ser Leu Tyr Tyr Met Ala Ala Ser Pro Lys Asp Pro Thr Tyr Val Val 260 265 270 Ala Pro Val Leu Lys Ser His Ser Gly Arg Glu Asp Leu Lys Lys Leu 275 280 285 Phe Ala Ala Trp Tyr Glu Arg Arg Gln Met Lys Lys Ile Tyr Ser Pro 290 295 300 Leu Leu Glu Ala Met Leu Ala Leu Tyr Leu Gly Phe Glu Trp Ile Gln 305 310 315 320 Thr Asn Asn Ile Phe Ala Pro Ile Ile Thr His Gly Ile Tyr Ser Ala 325 330 335 Val Ile Leu Gly His Gly Leu Trp Lys Ile His Asp His Arg Arg Arg 340 345 350 Leu His Gln Arg Ile Gln Gln Leu Lys Gln Glu Gly Asn Asn Ser Arg 355 360 365 Leo 370 <210> 31 <211> 224 <212> PRT <213> Nicotiana tabacum <400> 31 Met Gly Ala Gly Ile Pro Asp Glu Glu Glu Asn Asn Trp Pro Leu Trp 1 5 10 15 Leu Lys Pro Leu Leu Lys Glu Lys Phe Phe Gly His Cys Lys Leu His 20 25 30 Ala Asp Ser His Lys Ser Glu Cys Asn Met Tyr Cys Leu Asp Cys Ile 35 40 45 Asn Gly Pro Leu Cys Ser Leu Cys Leu Ala His His Lys Asp His Ile 50 55 60 Ala Ile Gln Ile Arg Arg Ser Ser Tyr His Asp Val Ile Arg Val Asn 65 70 75 80 Glu Ile Gln Lys Tyr Leu Asp Ile Ser Ser Val Gln Thr Tyr Ile Ile 85 90 95 Asn Ser Ala Lys Val Val Phe Leu Asn Glu Arg Pro Gln Pro Arg Pro 100 105 110 Gly Lys Gly Val Thr Asn Thr Cys Gln Val Cys Glu Arg Ser Leu Leu 115 120 125 Asp Ser Phe Lys Phe Cys Ser Leu Gly Cys Lys Val Val Gly Ser Ser 130 135 140 Lys Asn Phe Val Lys Lys Pro Lys Gln Leu Ser Ala Lys Arg Arg Arg 145 150 155 160 Ser Met Val Ala Ala Ser Asp Ser Asp Asp Ser Tyr Ser Ser Ser Ser 165 170 175 His Gly Arg Tyr Lys Ser His Ser Asn Lys Val Gln Ser Phe Thr Pro 180 185 190 Ser Thr Pro Pro Pro Thr Ser Val Asn Tyr Lys Thr Ala Lys Arg Arg 195 200 205 Lys Gly Ile Pro His Arg Ser Pro Met Gly Gly Leu Leu Ile Glu Tyr 210 215 220 <210> 32 <211> 452 <212> PRT <213> Nicotiana tabacum <400> 32 Met Ser Ser Asp Asn Phe Thr Asp Lys Asn Ala Val Phe Arg Lys Leu 1 5 10 15 Lys Ala Lys Pro Asp Asn Lys Met Cys Phe Asp Cys Asn Ala Lys Asn 20 25 30 Pro Thr Trp Ala Ser Val Thr Tyr Gly Ile Phe Leu Cys Ile Asp Cys 35 40 45 Ser Ala Thr His Arg Ser Leu Gly Val His Ile Ser Phe Val Arg Gly 50 55 60 Arg Cys Thr Val Arg Gly Thr Gly Leu Val Glu Leu Arg Ser Phe Gly 65 70 75 80 Leu Ser His Phe Leu Val Arg Asn His Tyr Leu His Pro Ile Cys Ala 85 90 95 Thr Thr Ala Ser Phe Cys Pro Thr Ile Gly Met Thr Thr Leu Arg Asp 100 105 110 Phe Met Cys Leu His Gln Gly Leu Trp Phe Ser Gly Lys Ser Ala Met 115 120 125 Phe Asp Val Trp Ile Arg Arg Met Pro Gln Ile Asp Lys Phe Arg Phe 130 135 140 Met Val Ser Arg Ala Val Lys Asp Asp Val Leu Trp Trp Lys Gln Pro 145 150 155 160 Cys Ser Ser Phe Phe Gln Ala Ala Leu Met Asp Gly Trp Arg Gln Glu 165 170 175 Asp Ala Gly Leu Pro Ala Ser Pro Phe Ala Ser Gln Ala Val Gln Ser 180 185 190 Thr Asn Gly Phe Ser Asp Val Lys Thr Ser Glu Ala Pro Lys Glu Thr 195 200 205 Ser Ser Phe Lys Glu Glu Thr Pro Ala Ser Pro Lys Ala Ser Gln Ser 210 215 220 Val Val Thr Thr Ser Ile Arg Lys Pro Ile Gly Ala Lys Lys Ser Gly 225 230 235 240 Lys Pro Gly Gly Gly Leu Gly Ala Arg Lys Leu Thr Lys Lys Pro Ser 245 250 255 Glu Ser Leu Tyr Asp Gln Lys Pro Glu Glu Pro Pro Val Gln Val Ser 260 265 270 Ser Ser Asn Ser Thr Ser Asn Ala Ser Thr Val Gly Ser Ser Phe Ala 275 280 285 Ser Arg Phe Glu Tyr Thr Asp Asn Val Gln Pro Ala Glu Met Ser Ser 290 295 300 Gly Gly Pro Arg Val Leu Asn His Val Ser Pro Pro Met Ser Ser Ser 305 310 315 320 Phe Phe Ser Asp Tyr Gly Met Asp Ser Gly Phe Thr Lys Lys Thr Ser 325 330 335 Ser Asn Ser Ser Lys Val Gln Ile Glu Glu Thr Asp Glu Ala Arg Lys 340 345 350 Lys Phe Ser Asn Ala Lys Ala Ile Ser Ser Ala Gln Phe Phe Gly Asp 355 360 365 Lys Ser Lys Ala Glu Met Glu Ala Ser Val Ser Leu Gln Lys Phe Ser 370 375 380 Gly Ser Ser Ala Ile Ser Ser Ala Asp Leu Phe Gly Asn Asp Asp Arg 385 390 395 400 Ala Asp Leu Asp Leu Thr Ala Gly Asp Leu Ile Asn Arg Leu Ser Phe 405 410 415 Gln Ala Gln Gln Asp Ile Ser Ser Leu Lys Asn Ile Ala Gly Glu Thr 420 425 430 Gly Lys Lys Leu Gly Ser Leu Ala Ala Thr Leu Met Ser Asp Phe Gln 435 440 445 Asp Arg Ile Leu 450 <210> 33 <211> 156 <212> PRT <213> Nicotiana tabacum <400> 33 Met Ala Ser Leu Ser Trp Trp Asn Pro Ala Pro Ala Thr Thr Ala Met 1 5 10 15 Ala Ala Cys Ser Pro Thr Pro Thr Ser Cys Lys Thr Ser Asn Ser Leu 20 25 30 Ala Leu Pro Arg Ser Val Phe Val Ser Lys Gln Ala Lys Leu Met Lys 35 40 45 Gln Ala Asn Gly Leu Leu Val Ile Thr Gln Gln Gln Ser Lys Lys Lys 50 55 60 Asn His Ser Phe Thr Asn Ser Arg Arg Asn Thr Ser Ile Gln Cys Leu 65 70 75 80 Ser Gln Glu Gln Lys Trp Thr His Glu Gly Ser Ile Thr Glu Ser Leu 85 90 95 Pro Asn Gly Met Phe Arg Val Lys Leu Asp Asn Ala Asp Val Val Leu 100 105 110 Gly Tyr Ile Ser Gly Lys Ile Arg Lys Asn Phe Ile Arg Leu Leu Pro 115 120 125 Gly Asp Arg Val Lys Ile Glu Val Ser Arg Tyr Asp Ser Thr Lys Gly 130 135 140 Arg Ile Ile Tyr Arg Leu Arg Gly Gly Arg Glu Gly 145 150 155 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 34 tgttagccct ttaccttggg ttt 23 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 35 tgtgattatc ggtgcaaaaa tgt 23 <210> 36 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic probe" <400> 36 aatggatcca gacaaac 17 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 37 cacagcaaca aggtccaaag ttt 23 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 38 tggccgtttt gtaattaact gaag 24 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic probe" <400> 39 actccgtcga cgccccctcc 20 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 40 ggcatctctc tcatggtgga a 21 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic primer" <400> 41 ggatgttgga gttggagaac aag 23 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic probe" <400> 42 cctgctcctg ccacgactgc aa 22 <210> 43 <211> 376 <212> PRT <213> Solanum lycopersicum <400> 43 Met Glu Phe Pro Leu Ile Ala Arg Cys Thr Asn Thr Pro Ser Thr Thr 1 5 10 15 Ser Phe Leu Gly Cys Lys Val Ser Leu Cys Asp Phe Pro Ile Arg Asn 20 25 30 Asn Tyr Arg Asp Lys Arg Asn Tyr Asn Glu Lys Phe Ser Val Val Arg 35 40 45 Ile Lys Ala Met Ala Glu Lys Ser Ser Thr Gly Glu Ala Ser Ser Val 50 55 60 Glu Ile Arg Glu Gly Glu Asn Gly Gly Val Gly Phe Thr Gly Ser Thr 65 70 75 80 Met Glu Val Thr Thr Phe Asn Gln Ser Phe Ser Asp Ala Gln Leu Pro 85 90 95 Val Trp Glu Lys Ile Gly Ala Val Val Arg Leu Ser Tyr Gly Ile Gly 100 105 110 Ile Tyr Gly Ala Met Ala Leu Ala Gly Lys Phe Ile Cys Ser Ile Ser 115 120 125 Gly Ile Asp Cys Thr Gly Gly Phe Ser Pro Ser Leu Asp Ala Ile Val 130 135 140 Glu Gly Leu Gly Tyr Ala Val Pro Pro Ile Met Ala Leu Leu Phe Ile 145 150 155 160 Leu Asp Asp Glu Val Val Lys Leu Ser Pro His Ala Arg Ala Ile Arg 165 170 175 Asp Val Glu Asp Glu Glu Leu Arg Asn Phe Phe Tyr Gly Met Ser Pro 180 185 190 Trp Gln...

Claims

1. 1. A modified tobacco plant or part thereof comprising a non-naturally occurring mutation in a polynucleotide having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18, 16, 17, 19-21 and 48, A modified tobacco plant or part thereof, wherein the modified tobacco plant comprises a buff phenotype, and wherein the mutation is relative to a control tobacco plant of the same tobacco variety.

2. 2. The modified tobacco plant or part thereof of claim 1, wherein the modified tobacco plant is of a tobacco variety selected from the group consisting of flue-cured, bright, Burley, Virginia, Maryland, dark, Galpao, Oriental, and Turkish varieties.

3. 2. The modified tobacco plant or part thereof of claim 1, wherein the modified tobacco plant is of a variety selected from the group consisting of the tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.

4. 2. The modified tobacco plant or part thereof of claim 1, which is heterozygous for the mutation.

5. 2. The modified tobacco plant or part thereof of claim 1, which is homozygous for the mutation.

6. 2. The modified tobacco plant or part thereof of claim 1, wherein the modified tobacco plant is a hybrid.

7. 2. The modified tobacco plant or part thereof of claim 1, wherein the modified tobacco plant is male sterile or cytoplasmic male sterile.

8. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-naturally occurring mutation results in a reduced expression level of the gene compared to a control tobacco plant.

9. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-naturally occurring mutation results in a reduced level of activity by a protein or polypeptide encoded by a polynucleotide comprising the non-naturally occurring mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-naturally occurring mutation.

10. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-naturally occurring mutation results in an increased expression level of the gene compared to a control tobacco plant.

11. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-naturally occurring mutation results in an increased level of activity by a protein or polypeptide encoded by a polynucleotide containing the non-naturally occurring mutation compared to a protein or polypeptide encoded by a polynucleotide lacking the non-naturally occurring mutation.

12. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-natural mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5' UTR, an intron, an exon, a 3' UTR, a terminator, and any combination thereof.

13. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-naturally occurring mutation comprises one or more mutation types selected from the group consisting of nonsense mutations, missense mutations, frameshift mutations, splice site mutations, and any combination thereof.

14. 2. The modified tobacco plant or part thereof of claim 1, wherein the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to a wild-type gene selected from the group consisting of SEQ ID NOs: 18, 16, 17, 19-21, and 48.

15. 2. The modified tobacco plant or part thereof of claim 1, wherein the tobacco plant comprises a mutation or transgene that confers reduced nicotine levels.

16. 16. The modified tobacco plant or part thereof of claim 15, wherein the tobacco plant is a low-alkaloid tobacco plant.

17. 16. The modified tobacco plant or part thereof of claim 15, wherein the mutation that confers reduced nicotine levels comprises a nic1 mutation, a nic2 mutation, or both.

18. 18. The modified tobacco plant or part thereof of claim 17, wherein the nic1 mutation, the nic2 mutation, or both, are introgressed from or derived from a variety selected from the group consisting of LA Burley 21, LAFC53, LN B&W, and LN KY171.

19. 16. The modified tobacco plant, or part thereof, of claim 15, wherein the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, an ethylene response factor (ERF) transcription factor, a nicotine uptake permease (NUP), and a MATE transporter.

20. 16. The modified tobacco plant or part thereof of claim 15, wherein the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus that encodes a protein selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2.

21. 16. The modified tobacco plant or part thereof of claim 15, wherein the mutation that confers reduced nicotine levels comprises a mutation in a gene or locus that encodes a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

22. 16. The modified tobacco plant or part thereof of claim 15, wherein the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBBl, BBL, MYC2, Nic1_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factors, nicotine uptake permease (NUP), and MATE transporters.

23. 16. The modified tobacco plant or part thereof of claim 15, wherein the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of ERF101, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2.

24. 16. The modified tobacco plant or part thereof of claim 15, wherein the transgene that confers reduced nicotine levels comprises a transgene that targets and represses a gene encoding a protein selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

25. 1. A modified tobacco plant or part thereof comprising a recombinant nucleic acid construct comprising a heterologous promoter operably linked to a polynucleotide encoding a non-coding RNA molecule, The modified tobacco plant or part thereof, wherein the non-coding RNA molecule is capable of binding to RNA encoding an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-33, 46, 47 and 50, the non-coding RNA molecule suppresses expression of the amino acid sequence, and the modified tobacco plant comprises a pale yellow phenotype.

26. 26. The modified tobacco plant or part thereof of claim 25, wherein the non-coding RNA molecule is a microRNA molecule.

27. 10. A dried tobacco material from the tobacco plant of claim 1.

28. 28. The dried tobacco material of claim 27, produced by a drying process selected from the group consisting of hot air drying, air drying, flame drying, and sun drying.

29. 28. A tobacco blend comprising the dry tobacco material of claim 27.

30. 30. The tobacco blend of claim 29, wherein the dried tobacco material comprises at least 10% by weight of the dried tobacco in the tobacco blend.

31. 30. The tobacco blend of claim 29, wherein the dried tobacco material comprises at least 10% by volume of the dried tobacco in the tobacco blend.

32. 28. A tobacco product comprising the dried tobacco material of claim 27.

33. 33. The tobacco product of claim 32, wherein the tobacco product is selected from the group consisting of cigarillos, non-breathable recess filter cigarettes, breathable recess filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarettes, chewing tobacco, leaf tobacco, hookah, shredded tobacco, and cut tobacco.

34. 33. The tobacco product of claim 32, wherein the tobacco product is a smokeless tobacco product.

35. 33. The tobacco product of claim 32, wherein the smokeless tobacco product is selected from the group consisting of loose-leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.

36. 28. A reconstituted tobacco comprising the dried tobacco material of claim 27.

37. 1. A method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, comprising: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and linked within 20 centimorgans (cM) of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising the PY QTL and the one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting a pale yellow phenotype. The method comprising:

38. 1. A method for producing a population of tobacco plants exhibiting a pale yellow (PY) phenotype, comprising: (a) genotyping a first population of tobacco plants or seeds for the presence of one or more marker loci associated with a PY quantitative trait locus (QTL) and located within 20,000,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5; (b) selecting one or more tobacco plants or seeds genotyped in step (a), wherein the one or more tobacco plants or seeds comprise the one or more marker loci and the PY QTL; and (c) producing a second population of tobacco plants or seeds comprising the PY QTL and the one or more marker loci from the one or more tobacco plants or seeds selected in step (b), wherein the second population of tobacco plants or seeds comprises at least one tobacco plant or seed exhibiting a pale yellow phenotype. The method comprising:

39. crossing the one or more tobacco plants produced in step (c) with a tobacco plant containing a mutation or transgene that directly suppresses or eliminates the expression or activity of one or more genes encoding products selected from the group consisting of methylputrescine oxidase (MPO), quinolate phosphoribosyltransferase (QPT), quinolinate synthase (QS), BBL, A622, aspartate oxidase, agmatine deiminase (AIC), S-adenosyl-methionine synthetase (SAMS), arginase, diamine oxidase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), arginine decarboxylase (ADC), nicotine uptake permease (NUP), and a MATE transporter.

39. The method of claim 37 or 38, further comprising:

40. 38. The method of claim 37, wherein the one or more marker loci are linked within 15 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

41. 38. The method of claim 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 10 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

42. 38. The method of claim 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

43. 38. The method of claim 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 1 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

44. 38. The method of claim 37, wherein the tobacco plant or tobacco seed of step (a) comprises a marker locus linked within 0.5 cM of a locus selected from the group consisting of SEQ ID NOs: 1-5.

45. 39. The method of claim 38, wherein the one or more marker loci are located within 500,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.

46. 39. The method of claim 38, wherein the one or more marker loci are located within 250,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.

47. 39. The method of claim 38, wherein the one or more marker loci are located within 100,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.

48. 39. The method of claim 38, wherein the one or more marker loci are located within 50,000 nucleotides of a locus selected from the group consisting of SEQ ID NOs: 1-5.

49. 39. The method of claim 37 or 38, wherein the buff phenotype comprises yellowing of at least 25% of the leaves of the progeny plant, on average at least 80%.

50. 39. The method of claim 37 or 38, wherein the locus is SEQ ID NO:

1.

51. 39. The method of claim 37 or 38, wherein the locus is SEQ ID NO:

2.

52. 39. The method of claim 37 or 38, wherein the locus is SEQ ID NO:

3.

53. 39. The method of claim 37 or 38, wherein the locus is SEQ ID NO:

4.

54. 39. The method of claim 37 or 38, wherein the locus is SEQ ID NO:

5.

55. 39. The method of claim 37 or 38, wherein the genotyping in step (a) comprises detecting one or more marker loci.

56. 56. The method of claim 55, wherein the one or more marker loci are selected from the group consisting of one or more single nucleotide polymorphism (SNP) markers, one or more insertion-deletion (INDEL) markers, one or more simple sequence repeat (SSR) markers, one or more restriction fragment length polymorphism (RFLP) markers, one or more random amplified polymorphic DNA (RAPD) markers, and one or more amplified fragment length polymorphism (AFLP) markers.

57. 56. The method of claim 55, wherein genotyping comprises the use of oligonucleotide probes.

58. 58. The method of claim 57, wherein the oligonucleotide probe comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34-42.

59. 58. The method of claim 57, wherein the oligonucleotide probe flanks a polymorphic nucleotide position of the marker locus.

60. 39. The method of claim 37 or 38, wherein the genotyping in step (a) comprises detecting haplotypes.

61. 61. The method of claim 60, wherein the haplotype comprises at least two SNPs selected from the group consisting of a guanine at nucleotide 121 of SEQ ID NO: 1, a guanine at nucleotide 121 of SEQ ID NO: 2, a guanine at nucleotide 101 of SEQ ID NO: 3, a thymine at nucleotide 121 of SEQ ID NO: 4, and a guanine at nucleotide 121 of SEQ ID NO:

5.

62. 39. The method of claim 37 or 38, wherein the tobacco plant is of a tobacco variety selected from the group consisting of Flue-Cured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish varieties.

63. 39. The method of claim 37 or 38, wherein the tobacco plant is of a variety selected from the group consisting of tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.

64. 39. The method of claim 37 or 38, wherein the one or more marker loci are selected from the group consisting of SEQ ID NOs: 1-5.

65. One or more marker loci are (a) guanine at nucleotide position 121 of SEQ ID NO: 1; (b) a guanine at nucleotide position 121 of SEQ ID NO: 2; (c) a guanine at nucleotide position 101 of SEQ ID NO: 3; (d) a thymine at nucleotide 121 of SEQ ID NO: 4; and (e) guanine at nucleotide 121 of SEQ ID NO: 5 65. The method of claim 64, wherein the single nucleotide polymorphism is selected from the group consisting of:

66. 1. A method for introgressing pale yellow (PY) QTL, comprising: (a) crossing a first tobacco plant containing the PY quantitative trait locus (QTL) with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) (i) a guanine at nucleotide 121 of SEQ ID NO: 1; (ii) a guanine at nucleotide 121 of SEQ ID NO: 2; (iii) a guanine at nucleotide 101 of SEQ ID NO: 3; (iv) a thymine at nucleotide 121 of SEQ ID NO: 4, or (v) guanine at nucleotide 121 of SEQ ID NO: 5 selecting a progeny plant or seed produced in step (a) that comprises at least one PY-associated single nucleotide polymorphism (SNP) selected from the group consisting of: The method comprising:

67. 1. A method for introgressing pale yellow (PY) trait, comprising: (a) crossing a first tobacco plant containing a non-naturally occurring mutation in a sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48 with a second tobacco plant of a different genotype to produce one or more progeny plants or seeds; and (b) selecting progeny plants or seeds produced in step (a) that contain the non-naturally occurring mutation, wherein the progeny plants or seeds contain the pale yellow trait. The method comprising:

68. 67. The method of claim 66, wherein the progeny plant or seed of step (b) is heterozygous for the SNP.

69. 67. The method of claim 66, wherein the progeny plants or seeds of step (b) are homozygous for the SNP.

70. 67. The method of claim 66, wherein the progeny plant or seed of step (b) comprises at least two SNPs selected from the group consisting of a guanine at nucleotide 121 of SEQ ID NO: 1, a guanine at nucleotide 121 of SEQ ID NO: 2, a guanine at nucleotide 101 of SEQ ID NO: 3, a thymine at nucleotide 121 of SEQ ID NO: 4, and a guanine at nucleotide 121 of SEQ ID NO:

5.

71. 67. The method of claim 66, wherein the single nucleotide polymorphism is the guanine at nucleotide 121 of SEQ ID NO:

1.

72. 67. The method of claim 66, wherein the single nucleotide polymorphism is the guanine at nucleotide 121 of SEQ ID NO:

2.

73. 67. The method of claim 66, wherein the single nucleotide polymorphism is the guanine at nucleotide 101 of SEQ ID NO:

3.

74. 67. The method of claim 66, wherein the single nucleotide polymorphism is a thymine at nucleotide 121 of SEQ ID NO:

4.

75. 67. The method of claim 66, wherein the single nucleotide polymorphism is the guanine at nucleotide 121 of SEQ ID NO:

5.

76. 68. The method of claim 66 or 67, wherein the tobacco plant is of a tobacco variety selected from the group consisting of Flue-Cured, Bright, Burley, Virginia, Maryland, Dark, Galpao, Oriental, and Turkish varieties.

77. 68. The method of claim 66 or 67, wherein the tobacco plant is of a variety selected from the group of tobacco varieties set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.

78. 68. The method of claim 67, wherein the progeny plant or seed is heterozygous for the non-naturally occurring mutation.

79. 68. The method of claim 67, wherein the progeny plant or seed is homozygous for the non-naturally occurring mutation.

80. 68. The method of claim 67, wherein the non-naturally occurring mutation results in a reduced expression level of the gene compared to a control tobacco plant.

81. 68. The method of claim 67, wherein the non-naturally occurring mutation results in an increased expression level of the gene compared to a control tobacco plant.

82. 68. The method of claim 67, wherein the non-naturally occurring mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5' UTR, an intron, an exon, a 3' UTR, a terminator, and any combination thereof.

83. 68. The method of claim 67, wherein the non-naturally occurring mutation 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.

84. 68. The method of claim 67, wherein the non-naturally occurring mutation comprises a mutation selected from the group consisting of one or more nucleotide substitutions, deletions, insertions, duplications, and inversions relative to a wild-type nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16-21 and 48.

85. 67. The method of claim 66, wherein the progeny plant or seed comprises a reduced level of at least one tobacco-specific nitrosamine (TSNA) compared to the second tobacco plant lacking the PY QTL.

86. 86. The method of claim 85, wherein the at least one TSNA is selected from the group consisting of N-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone, N'-nitrosoanatabine, and N'-nitrosoanabasine.

87. 67. The method of claim 66, wherein the progeny plant or seed comprises an increased USDA leaf grade index compared to the second tobacco plant lacking the PY QTL.

Images