Low-reverting tobacco plants with low nornicotine content, selection method thereof, and method for producing tobacco plant lines

Abstract

Provided are a method for producing a low-reversion type tobacco plant line having low nornicotine content without using genetic engineering and artificial mutagenesis techniques, and a tobacco plant. The method according to the present invention comprises identifying a variety-specific DNA sequence linked to the low-reversion type and low nornicotine traits and making selection with the use of this sequence as an indicator.

Description

[Technical Field]

[0001] The present invention relates to a tobacco plant having a low revertant low nornicotine content, a method for selecting the same, and a method for producing a tobacco plant line. [Background technology]

[0002] Nornicotine is a precursor of N-nitrosonornicotine (NNN), a tobacco-specific nitrosamine (TSNA). The ability to stably suppress nornicotine at low levels is important for tobacco breeding. NNN is produced by the non-enzymatic reaction of nornicotine with nitrite and other substances after nicotine is converted to nornicotine by nicotine N-demethylase (NND). NND genes form a gene family, and three NNDs (CYP82E4, CYP82E5, and CYP82E10) are functional in Nicotiana tabacum. Of these, the major gene encodes CYP82E4, which is induced in senescent (mature) and drying leaves (Non-Patent Document 1), and conversion by CYP82E4 accounts for 98% of nornicotine production. CYP82E4 in the amphidiploid Nicotiana tabacum is encoded by the T-type genome, one of the two genomes present in Nicotiana tabacum: the S-type genome derived from Nicotiana sylvestris and the T-type genome derived from Nicotiana tomentosiformis. Tobacco breeding methods focusing on NND gene expression have been reported, including low-nornicotine breeding using CYP82E4 gene mutations as an indicator (Patent Document 1), RNAi of the NND gene (Non-Patent Document 2), and selection of EMS (ethylmethane sulfonate) tobacco knockout mutants of the NND gene (Non-Patent Documents 3 and 4). It is also known that the promoter region of the CYP82E4 NND gene contains 12 guanine-adenine (GA) repeats (Patent Documents 1 and 2). [Prior art documents] [Chartered documents]

[0003]

Patent Document 1

Patent document 2

Non-licensed literature

[0004]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

[0005] Tobacco plants used as raw materials for tobacco products are often selected for their low nornicotine content (so-called low-converter varieties). However, with each generation, individuals with high nornicotine content (hereinafter also referred to as high-nornicotine converters) emerge at a certain frequency, which presents a problem. However, the cause of the emergence of such individuals is not well understood. On the other hand, because the above-mentioned techniques for producing tobacco with reduced NNN content manipulate the structural gene itself, it is thought that high-nornicotine converters are unlikely to emerge in RNAi and knockout mutants of the CYP82E4 gene even after multiple generations. However, obtaining these tobacco plants requires the use of genetic engineering or artificial mutagenesis techniques, and the use of mutagenesis techniques may result in unwanted background mutations in the resulting individuals.

[0006] One aspect of the present invention aims to enable the creation of low-revertant, low-nornicotine content tobacco varieties that are less likely to produce high-nornicotine type convertors even after successive generations, based on conventional breeding methods, without using genetic engineering technology or artificial mutant production technology. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors conducted extensive research and discovered that Matsukawa (Kanto), a tobacco variety native to Japan, is a variety in which high-nornicotine convertors are unlikely to appear even after generations. Next, they discovered that this low-revertant, low-nornicotine trait is linked to genomic DNA sequences specific to Matsukawa (Kanto), and that these DNA sequences can be used as indicators to select tobacco plants with low revertant, low-nornicotine content.

[0008] That is, in one aspect of the present invention, a low-reverting, low-nornicotine tobacco plant is selected from a breeding progeny obtained by crossing the Nicotiana tabacum cultivar Matsukawa (Kanto) with another Nicotiana tabacum cultivar or its mutant, and the low-reverting, low-nornicotine trait is linked to a genomic DNA sequence specific to Matsukawa (Kanto).

[0009] In one embodiment of the present invention, a low-revertant, low-nornicotine tobacco plant having an endogenous CYP82E4 gene encoding the amino acid sequence set forth in SEQ ID NO:2 is selected. [Effects of the Invention]

[0010] According to one aspect of the present invention, it is possible to create tobacco varieties with low revertant, low nornicotine content without using genetic engineering techniques and without the unwanted background mutations that are problematic when using mutants. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of an analysis of the nornicotine ratio of a crossbred progeny (BC5F2) line between Matsukawa (Kanto) and TN90 in an example. DETAILED DESCRIPTION OF THE INVENTION

[0012] The following describes in detail the embodiments of the present invention. Note that the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0013] [Method for selecting tobacco plants with low revertant and low nornicotine content] One embodiment of the present invention provides a method for selecting a low-reverting, low-nornicotine tobacco plant from a breeding progeny obtained by crossing Matsukawa (Kanto), a Japanese native species of Nicotiana tabacum, with another Nicotiana tabacum variety or its mutant.

[0014] As used herein, " low reversion type " refers to the trait that individuals with high nornicotine are unlikely to appear even after crossbreeding within a variety and passing through generations.Specifically, it refers to the trait that the number of individuals whose nornicotine ratio, that is, the ratio of nornicotine content to the total of nornicotine content and nicotine content, is more than 5% within a variety or line, is less than 10%, preferably less than 5%, or the trait that the number of individuals whose nornicotine ratio is more than 10% within a variety or line, is less than 5%, preferably less than 2%.

[0015] "Japanese native varieties" is a general term for tobacco varieties cultivated in various parts of Japan before the introduction of flue-cured and burley varieties, as well as tobacco varieties that have been improved based on them. Matsukawa (Kanto) is a Japanese native variety and a milder variety for cigarettes. The Matsukawa variety includes many varieties, including Matsukawa (Kanto) and Matsukawa (Fukushima). When simply referred to as "Matsukawa," it generally refers to "Matsukawa (Fukushima)" rather than "Matsukawa (Kanto)." As shown in the examples, "Matsukawa (Kanto)" and "Matsukawa (Fukushima)" can be distinguished using the markers described below. The following three varieties are registered as Matsukawa in the USDA (United States Department of Agriculture) accession list: Kanto (Matsukawa): TI 1587, Kanto 201 (Matsukawa): TI 1588, Matsukawa: TI 168. Of these, "Kanto (Matsukawa): TI 1587" is "Matsukawa (Kanto)."

[0016] The other Nicotiana tabacum cultivar to be crossed with Matsukawa (Kanto) may be any Nicotiana tabacum cultivar other than Matsukawa (Kanto), including, but not limited to, burley, flue-cured, and native Japanese varieties. The other cultivar may be either a cultivar with a relatively low nicotine-to-nornicotine conversion rate (hereinafter also referred to as low-converter) or a cultivar with a relatively high nicotine-to-nornicotine conversion rate (hereinafter also referred to as high-converter). Even in low-converter cultivars, high-nornicotine converters are frequently observed, and thus seeds are selected for nornicotine content for commercial use. Furthermore, in the case of high-converter cultivars, almost all individuals are high-nornicotine converters. The other cultivar may also be a mutant with a desired mutation in its genome that confers specific traits to the individual.

[0017] A method for selecting tobacco plants with a low-reverting, low-nornicotine content comprises selecting individuals that are homozygous for one or more markers in the genome sequence that are linked to the low-reverting, low-nornicotine trait. The markers are markers based on single-base substitutions, insertions, or deletions corresponding to specific positions in the genome sequence of Nicotiana tabacum. The markers used in the method of this embodiment are listed in Tables 1 and 2 below. The markers are described below.

[0018] [Table 1]

[0019] [Table 2]

[0020] Table 1 shows genomic markers based on single base substitutions (hereinafter referred to as SNV markers). Table 2 shows genomic markers based on insertions or deletions (hereinafter referred to as InDel markers). The genomic locations of each marker shown in Tables 1 and 2 are shown as positions on chromosome Nt09 of the reference genome sequence of Nicotiana tabacum cultivar K326. The reference genome sequence of K326 referred to herein is the sequence constructed by Edwards et al., 2017. BMC Genomics, 18(1), 1-14 (DOI: 10.1186 / s12864-017-3791-6) and published on the Sol Genomics Network (https: / / solgenomics.net / ), and is the sequence published at the following URL: ftp: / / ftp.solgenomics.net / genomes / Nicotiana_tabacum / edwards_et_al_2017 / assembly / Nitab-v4.5_genome_Chr_Edwards2017.fasta (last updated: April 13, 2017).

[0021] The "base" shown in Table 1 indicates the type of base at the position corresponding to the "position on Nt09" in the genome sequence of Matsukawa (Kanto). The markers shown in Table 1 are markers that can distinguish Matsukawa (Kanto) from at least one of K326, a representative flue-cured variety, or TN90, a representative burley variety.

[0022] Table 3 below shows the bases at each SNV marker site in K326, TN90, and Matsukawa (Kanto). In Table 3, the TN90 base at each marker site was determined with reference to publicly available sequence data (accession no. SRR954964 in GenBank's Sequence Read Archive (SRA)). Furthermore, the K326 base at each marker site was determined not based on the K326 reference genome sequence described above, but with reference to other publicly available sequence data (accession no. SRR955769 in GenBank's Sequence Read Archive (SRA)). Note that, in this specification, the type of base at each marker site in each variety, or the presence or absence of deletion or insertion, or the state thereof, is also referred to as the "genotype" of that variety.

[0023] For example, the corresponding base for SNV1 is "C" in Matsukawa (Kanto), and "T" in K326 and TN90. Therefore, this marker can be used to distinguish between the Matsukawa (Kanto) genotype and the K326 and TN90 genotypes. Furthermore, the corresponding base for SNV3 is "C" in Matsukawa (Kanto), "C" in K326, and "T" in TN90. Therefore, this marker can be used to distinguish between the Matsukawa (Kanto) genotype and the TN90 genotype. Furthermore, the corresponding base for SNV6 is "G" in Matsukawa (Kanto), "A" in K326, and "G" in TN90. Therefore, this marker can be used to distinguish between the Matsukawa (Kanto) genotype and the K326 genotype.

[0024] [Table 3]

[0025] The "insertion / deletion" in Table 2 indicates the presence or absence of an insertion or deletion at a position corresponding to the "position on Nt09" in the genome sequence of Matsukawa (Kanto) in comparison with the reference genome. The markers in Table 2 are markers that can distinguish Matsukawa (Kanto) from at least one of the flue-cured rice variety K326 or the burley variety TN90. In the "position on Nt09" in Table 2, a notation indicated by "^", for example, "101544774^101544775", indicates (the presence or absence of) an insertion between bases 101544774 and 101544775 of Nt09 in the reference genome. A notation indicated by ".", for example, "102040788..102040807", indicates (the presence or absence of) a deletion between bases 102040788 and 102040807 of Nt09 in the reference genome.

[0026] Table 4 below shows the status of each InDel marker site in K326, TN90, and Matsukawa (Kanto). Note that the genome sequence information for K326 and TN90 in Table 4 also references the same data as in Table 3. For example, in the corresponding position of InDel1, an insertion of the base "A" is observed in Matsukawa (Kanto), while neither an insertion nor a deletion is observed in K326 or TN90. Therefore, this marker can be used to distinguish the genotype of Matsukawa (Kanto) from the genotypes of K326 and TN90. Furthermore, in the corresponding position of InDel3, an insertion of the base "TT" is observed in Matsukawa (Kanto) and TN90, while a "T" insertion is observed in K326. Therefore, this marker can be used to distinguish the genotype of Matsukawa (Kanto) from the genotype of K326. Furthermore, at the corresponding position of InDel9, a "T" base insertion is observed in Matsukawa (Kanto) and K326, while a "TT" base insertion is observed in TN90. Therefore, this marker can be used to distinguish the genotypes of Matsukawa (Kanto) and TN90. The insertion of a base in K326 is due to a discrepancy between the sequence information of the reference genome used to identify the position on Nt09 and the sequence data used to identify the genotype of K326.

[0027] [Table 4]

[0028] As mentioned above, some markers are identical to the genotypes of flue-cured rice or burley-cured rice. Therefore, the markers used are selected from the table above and are distinguishable from the genotypes of the other varieties to be crossed. For example, when a flue-cured rice variety is used as the other variety to be crossed, markers that can distinguish Matsukawa (Kanto) from flue-cured rice can be selected and used during selection. When a burley-cured rice variety is used as the other variety to be crossed, markers that can distinguish Matsukawa (Kanto) from burley can be selected and used during selection. In addition, the genotype of each marker in the other variety to be crossed may be examined in advance to confirm whether the markers are appropriate.

[0029] Methods for detecting markers include, but are not limited to, (1) a method in which a DNA sequence containing the marker is amplified by PCR or the like, and then the DNA base sequence is directly decoded using a commercially available sequencer, (2) a method in which sequence differences are detected by the difference in electrophoretic distance using the SSCP (Single Strand Conformation Polymorphism) method, (3) a method in which SNPs (Single Nucleotide Polymorphisms) are detected using the Cycleave PCR method, (4) a method in which the presence or absence of mutations is detected by cleaving mismatch sites using T7 Endonuclease I or the like, (5) the CAPS (Cleaved Amplified Polymorphic Sequence) method, which can determine the presence or absence of mutations based on the presence or absence of cleavage by restriction enzyme treatment, and (6) the dCAPS (Derived Capsid Sequence Sequence) method, which can determine the presence or absence of mutations based on the presence or absence of cleavage by restriction enzyme treatment using a primer set that intentionally contains mismatches. (7) a method for determining the presence or absence of a mutation by detecting whether or not a probe that specifically hybridizes to a mutant sequence has hybridized (PCR using TaqMan probes); (8) a method for performing single-base extension using a primer adjacent to the mutation and detecting the presence or absence of a mutation based on the difference in mass of the incorporated base (MassARRAY analysis); and (9) a method for detecting mutations based on differences in electrophoretic mobility in the case of deletions or insertions. Any method that can determine the presence or absence of a mutation will suffice, depending on the type of mutation used as a marker.

[0030] Although not limited to, for example, in the case of InDel markers, a method can be preferably used in which a short PCR product containing the sequence is amplified and products containing the insertion or deletion are electrophoretically distinguished from products that do not, and in the case of SNV markers, a method can be preferably used in which a restriction enzyme is used and electrophoresis is used to distinguish them (CAPS method).

[0031] The number of markers used may be one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or may be 11 or more.

[0032] The markers to be used are not particularly limited, but preferably include any of InDel4, SNV1, and SNV95.

[0033] A method for confirming that a tobacco plant has a low-nornicotine trait is, for example, a nornicotine assay using isatin staining. The assay includes the steps of aging tobacco plant seedlings to promote the conversion of nicotine to nornicotine, extracting nornicotine from a portion of the leaf and reacting it with isatin, and confirming the presence or absence of the nornicotine trait based on the degree of color development of blue spots. Other methods for confirming the low-nornicotine trait include, for example, gas chromatography (GC) or gas chromatography mass spectrometry (GC-MS).

[0034] [Method for producing a tobacco plant line with low revertant and low nornicotine content] The present invention also provides a method for producing a low-reverting, low-nornicotine tobacco plant line using the above-mentioned selection method, and a tobacco plant obtained by the method. The production method according to one embodiment of the present invention includes a first step of crossing a Nicotiana tabacum variety, Matsukawa (Kanto), with another Nicotiana tabacum variety or its mutant to obtain a first-generation hybrid plant (hereinafter also referred to as F1), a second step of repeatedly backcrossing the first-generation hybrid plant, selecting individuals that have the marker as a heterozygote after each backcrossing and using them in the next backcrossing, and a third step of self-pollinating the individuals obtained as a result of the second step to obtain breeding progeny, and then performing the selection method to select individuals that have the marker as a homozygote.

[0035] The Nicotiana tabacum cultivar or mutant to be crossed with Matsukawa (Kanto) in the first step is an individual to which the low-reverting, low-nornicotine trait possessed by Matsukawa (Kanto) is to be imparted, and there are no particular restrictions as long as it is a cultivar that can be crossed with Matsukawa (Kanto). Crossing and selection of F1 plants can be performed according to conventional methods. Since all F1 plants are heterozygous for the genomic structure of Matsukawa (Kanto) that is involved in imparting the low-reverting, low-nornicotine trait, any F1 plants can be used in the second step.

[0036] In the second step, the first-generation hybrid plants obtained in the first step are backcrossed. The parent used for backcrossing is typically the same as that used in the first step, but it can also be a different Nicotiana tabacum variety. For example, if an EMS-treated mutant was used in the first step, a non-EMS-treated individual with the same background can be used for backcrossing instead. Alternatively, to confer additional superior traits, a variety with other superior traits and cultivated for commercial purposes (so-called "elite varieties") can be used for backcrossing.

[0037] In backcrossing, individuals heterozygous for the Matsukawa (Kanto) genome structure, which is related to the conferring of the low-reverting, low-nornicotine trait, and individuals without this structure appear in a theoretical ratio of 1:1. Therefore, to select individuals with the Matsukawa (Kanto) genome structure, which is related to the conferring of the low-reverting, low-nornicotine trait, one or more markers selected from Tables 1 and 2 that can distinguish the different cultivars used in the first crossing and the cultivars used in the backcrossing in the second crossing are used to select individuals heterozygous for the Matsukawa (Kanto) genotype for the markers after each backcrossing. Then, individuals heterozygous for the Matsukawa (Kanto) genotype for the markers are used in the next backcrossing. Backcrossing is performed repeatedly in this manner, but there is no limit to the number of times; for example, backcrossing can be performed three to six times. For confirmation, the nornicotine content or the NN-index, as described below, may be measured for individuals heterozygous for the Matsukawa (Kanto) genotype selected based on the marker genotype. Furthermore, based on the results of measuring nornicotine content or confirming the NN-index described below, further selection of individuals with a lower nornicotine content may be carried out for individuals with the Matsukawa (Kanto) genotype selected based on the marker genotype.

[0038] In the third step, the individuals finally obtained in the second step, for example, individuals obtained by performing three to six backcrosses, are selfed to select individuals homozygous for the Matsukawa (Kanto) genotype for the marker. The method for selecting individuals homozygous for the Matsukawa (Kanto) genotype for the marker can be the method for selecting low-reverting, low-nornicotine-content tobacco plants described above. This allows for the creation of low-reverting, low-nornicotine-content tobacco plant lines derived from tobacco plants with a desired background. For confirmation, the tobacco plants selected based on the marker genotype may be subjected to measurement of nornicotine content or confirmation of the NN-index described below. Furthermore, based on the results of measurement of nornicotine content or confirmation of the NN-index described below, further selection of individuals with lower nornicotine content may be performed on the low-reverting, low-nornicotine-content tobacco plants selected based on the marker genotype.

[0039] That is, one embodiment of the present invention relates to tobacco plants with low revertant and low nornicotine content obtained by the above-described production method, and their progeny. Therefore, these plants are not Matsukawa (Kanto) plants themselves, but they possess the low revertant and low nornicotine trait of Matsukawa (Kanto). These plants are characterized by having an endogenous CYP82E4 gene encoding the amino acid set forth in SEQ ID NO: 2 and by having, as homozygotes, one or more markers selected from Tables 5 and 6 below. The markers listed in Tables 5 and 6 are those listed in Tables 1 and 2 that can distinguish the plants from both flue-cured K326 and burley TN90.

[0040] [Table 5]

[0041] [Table 6]

[0042] 〔summary〕 By summarizing the above embodiments, the present invention can be summarized as follows.

[0043] (1) A method for selecting tobacco plants with low revertant, low nornicotine content, comprising the step of selecting individuals in a breeding progeny line obtained by crossing the Nicotiana tabacum variety Matsukawa (Kanto) with another Nicotiana tabacum variety or a mutant thereof, which have, as homozygotes, one or more markers of the variety Matsukawa (Kanto), which are selected from Tables 1 and 2 and which distinguish the plant from the other variety or its mutant.

[0044] (2) The selection method according to (1), wherein the different variety is a burley variety or a flue-cured variety.

[0045] (3) The selection method according to (1) or (2), wherein the one or more markers include at least one of InDel4, SNV1, and SNV95.

[0046] (4) The selection method according to (1) or (2), wherein the one or more markers include at least InDel4.

[0047] (5) A first step of crossing a Nicotiana tabacum variety, Matsukawa (Kanto), with another Nicotiana tabacum variety or its mutant to obtain a first generation hybrid plant; a second step of repeatedly backcrossing the first generation hybrid plants with a Nicotiana tabacum other than Matsukawa (Kanto) as a backcross parent, wherein after each backcrossing, individuals having as heterozygotes one or more markers of Matsukawa (Kanto) selected from the table that distinguish them from the other cultivar or mutant and the backcross parent are selected and used in the next backcrossing; A method for producing a tobacco plant line with a low revertant, low nornicotine content, comprising a third step of self-pollinating the individuals obtained as a result of the second step to obtain breeding progeny, and then carrying out the selection method described in (1) to select individuals that have one or more of the above markers as homozygotes.

[0048] (6) A tobacco plant with a low revertant, low nornicotine content, obtained by the production method described in (5).

[0049] (7) Having an endogenous CYP82E4 gene encoding the amino acid sequence shown in SEQ ID NO: 2; A tobacco plant with a low revertant, low nornicotine content, which has one or more markers selected from Tables 5 and 6 as homozygotes (excluding Matsukawa (Kanto), a variety of Nicotiana tabacum).

[0050] The following examples are provided to further explain the embodiments of the present invention. Of course, the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference. [Example]

[0051] (1) Tobacco plants The cultivars used in this example were Matsukawa (Kanto), a native Japanese tobacco; TN90 and Burley 21 (No. 3-10), flue-cured K326, Tsukuba 1, and the Tsukuba 1 mutant. TN90, K326, and Tsukuba 1 are low-converter tobaccos with a low rate of nicotine to nornicotine conversion, while Burley 21 (No. 3-10) is a high-converter tobacco with a high rate of nornicotine conversion. Furthermore, an F2 segregating population was produced by selfing F1 seeds obtained by crossing Matsukawa (Kanto) with Burley 21 (No. 3-10), and the resulting F2 seeds were used for analysis. Plants were grown in a greenhouse.

[0052] (2) Low-reverting low-nornicotine trait in Matsukawa (Kanto) 226 Matsukawa (Kanto) individuals were grown in a greenhouse and subjected to a nornicotine assay. The nornicotine assay was performed using a method that combined seedling senescence induction (Shi et al. 2003, J. Agric. Food Chem. 51: 7679-7683.) and isatin staining (Sato and Asane 1982, Iwate Experimental Report 14:17-21.). Seedlings 6-7 weeks after sowing (3-week-old seedlings were temporarily transplanted into 4x4 vinyl pots and then 3-4 weeks old seedlings) grown in a greenhouse were treated with aging treatment at 37°C and 85% humidity for 4 days to promote the conversion of nicotine to nornicotine. Afterwards, a portion of the leaf (approximately 1.5 cm from the tip) was sown. 2 The cells were placed in a 1.5-mL Eppendorf tube and immersed in 250 μL of a strong alkaline solution (8 M NaOH) containing 1% Tween-20 surfactant to disrupt the cells. Then, 500 μL of chloroform was added and the mixture was left to stand for 1 hour to extract nornicotine. 20 μL of the lower chloroform layer was spotted onto filter paper (Absorbent paper CB-09A, ATTO). After the solution dried, isatin reagent [0.1 g of 2,3-indolinedione (Wako) dissolved in 2.5 mL of acetic acid and 50 mL of ethanol] was sprayed evenly onto the filter paper using a glass sprayer and then heated in a hot air oven at 120°C for 4 minutes. The reaction between isatin and nornicotine produces a blue spot. As an indicator of color development, standard nornicotine was similarly dropped to create indicators with a five-level index (NN-index = 1 to 5) corresponding to the degree of color development. Specifically, indicators were created with five levels of standard nornicotine content (0.05%, 0.1%, 0.25%, 0.5%, and 1%) as indicators of color development, and these were used as a reference for the degree of color development. These color developments were designated as nornicotine index values ​​(or nornicotine indices) of 1, 2, 3, 4, and 5, respectively. Using this indicator, the NN-index was determined from the degree of color development. In this indicator, the higher the NN-index, the higher the nornicotine content.

[0053] The results of the nornicotine assay for each line are shown in Table 7. In the control Burley 21 (No. 3-10), all 23 individuals had an NN-index of 3 or higher, with nearly 70% exhibiting an NN-index of 4 or 5. In other words, all Burley 21 (No. 3-10) individuals had high nornicotine content and were typical high converters. In TN90, 30 of 48 individuals had an NN-index of 1 or 2, indicating low nornicotine content. However, 9 individuals had an NN-index of 4 or 5, indicating the emergence of high nornicotine converters. In contrast, all 226 individuals in Matsukawa (Kanto) had a low nornicotine content, indicating an NN-index of 1 or 2, and no individuals with an NN-index of 3 or higher were observed. These results confirmed that Matsukawa (Kanto) is a tobacco variety in which high nornicotine converters do not or are extremely unlikely to emerge.

[0054] [Table 7]

[0055] (3) CYP82E4 gene mutation in Matsukawa (Kanto) Next, the nucleotide sequence of the CYP82E4 gene of Matsukawa (Kanto) was determined based on the known genomic sequence of the CYP82E4 gene of TN90 (JP Patent Publication No. 2008-506362, SEQ ID NO: 4, and GenBank accession number: AYMY01253998). The sequences of the same gene in TN90, Burley 21 (No. 3-10), and K326 were compared. Genomic DNA was extracted from leaves of Matsukawa (Kanto) seedlings using the Gentra Puregene Tissue Kit (Qiagen). Using this DNA as a template, the first half (PCR1) and second half (PCR2) of the CYP82E4 gene (approximately 6500 bp) were amplified by PCR, and the sequence was determined. PCR was performed using 5 ng of template DNA and 5 pmol of each of the primers listed in Table 8 in a 20 μL reaction mixture using Tks (GFlex) DNA Polymerase (Takara Bio Inc.), with 40 cycles of 96°C for 10 seconds, 55°C for 10 seconds, and 72°C for 15 seconds. The PCR products (approximately 3.1 kb from PCR1 and approximately 3.4 kb from PCR2) were treated with ExoSAP-IT (Thermo Fisher Scientific) to remove the PCR primers, and then sequenced using a fluorescent sequencer. The entire CYP82E4 gene sequence was then determined using the primer walk method.

[0056] [Table 8]

[0057] The control strain, TN90, contained 12 GA repeats in the promoter region approximately 500 bp upstream of the ATG translation initiation codon of the CYP82E4 gene, whereas Matsukawa (Kanto) contained a four-base "GAGA" insertion (hereafter also referred to as a GAGA insertion). This resulted in 14 GA repeats in the promoter of the CYP82E4 gene in Matsukawa (Kanto). No mutations were found in other regions. On the other hand, a similar experiment was performed on Burley 21 (No. 3-10), revealing 12 GA repeats. Furthermore, a search of the CYP82E4 genome sequence of K326 published in a public database revealed 12 GA repeats. The GA repeats in Tsukuba 1 were also 12. Furthermore, confirmation of Matsukawa (Fukushima) revealed that Matsukawa (Fukushima) contained 13 GA repeats, confirming its distinct genomic sequence from Matsukawa (Kanto).

[0058] (4) Obtaining mutants of the CYP82E4 gene (experimental control) Tobacco mutants with a disrupted CYP82E4 gene were obtained as experimental controls. From the Tsukuba No. 1 mutant library (Tajima et al. 2011 Ann. Phytopathol. Soc. Jpn. 77:258., Takakura et al. 2018 Mol. Plant Pathol. 19 2124-2133.), a mutant with a nonsense mutation in the first exon of the CYP82E4 gene (SEQ ID NO: 4 in JP 2008-506362 A) was selected (G → A substitution at base 2798 of SEQ ID NO: 1 in the Sequence Listing, resulting in a TGG-TGA codon). After obtaining an amplified product using the primers listed in Table 8 (for PCR1), the base sequence was determined using the mutation detection primers (Table 9) to obtain a homozygous mutant line. The resulting mutant line was designated e4-TUM.

[0059] [Table 9]

[0060] (5) Confirmation of the inheritance mode of the low-reverting low-nornicotine trait in Matsukawa (Kanto) and its linkage to the GAGA insertion As mentioned above, Matsukawa (Kanto) showed that high-nornicotine convertors did not or were extremely unlikely to appear. To investigate whether the genetic factors responsible for the low-revertant, low-nornicotine trait in Matsukawa remain effective when crossed with other cultivars and whether low-nornicotine individuals could be selected using the GAGA insertion as an indicator, we analyzed the linkage between the GAGA insertion and nornicotine type using an F2 segregating population of the progeny of a cross between Matsukawa (Kanto) and Burley 21 (No. 3-10). The presence or absence of the GAGA insertion was confirmed by PCR using the same primers (Forward primer 1 and Reverse primer 1) as Target PCR1 in Table 8, as described above, with DNA extracted from leaves of seedlings 7 weeks after sowing as a template. The nucleotide sequence of the amplified product was determined using the sequencing primers near the GAGA insertion in Table 10. The GAGA insertion was identified as homozygous, heterozygous, or null using the waveform of the sequence data.

[0061] [Table 10]

[0062] Table 11 shows the results of nornicotine assays for the F2 segregating population of the cross between Matsukawa (Kanto) and Burley 21 (No. 3-10) and the control cultivar. Of the 49 individuals homozygous for the GAGA insertion, 47 had an NN index of 1 and 2 had an NN index of 2; none had an NN index of 3 or higher. This was similar to the results for the control tobacco mutant line (e4-TUM) with a nonsense mutation in the CYP82E4 gene. In contrast, in the populations heterozygous for the GAGA insertion or without the GAGA insertion, although some individuals had an NN index of 1 or 2, most had an NN index of 3 or higher. Furthermore, in the control cultivars TN90 and Tsukuba 1 (both seed-selected), many individuals had an NN index of 1 or 2. However, 2 of 47 TN90 individuals and 6 of 48 Tsukuba 1 individuals had an NN index of 4 or higher, indicating the emergence of high-nornicotine converters. Furthermore, the pattern of emergence of high-nornicotine converters in TN90 (appearance in 2 out of 47 individuals) was similar to that of TN90LC in Verrier et al. (2011) (CORESTA Meeting AP06), and the pattern of emergence of high-nornicotine converters in Tsukuba 1 (appearance in 6 out of 47 individuals) was similar to that in Kubo (1985) (Iwata Tobacco Research Station Report 17:69-137). From the above, it became clear that the GAGA insertion in Matsukawa (Kanto) is linked to the low-revertant, low-nornicotine trait, and that tobacco plants with low-revertant, low-nornicotine content can be selected using the GAGA insertion as an indicator.

[0063] [Table 11]

[0064] The appearance of NN-index = 1 or 2 in heterozygous and null (no GAGA insertion) plants in the F2 population may have been due to low accumulation of nicotine, the substrate for nornicotine. Therefore, all 58 plants with NN-index = 1 or 2 in the F2 population (Table 11), as well as five control plants in TN90 with an NN-index = 1, were transplanted from the vinyl pots to No. 4 pots to promote nicotine biosynthesis, and a nornicotine assay was performed three weeks later. Four weeks after transplanting, all heterozygous and null plants with an NN-index = 1 or 2 for the GAGA insertion had an NN-index = 4 or 5 three weeks after transplanting. Furthermore, all plants homozygous for the GAGA insertion that had an NN-index = 1 or 2 remained at an NN-index = 1 or 2. As a result, all plants homozygous for the GAGA insertion had low nornicotine content, and no high-nornicotine conversion mutants were observed. On the other hand, of the five individuals with an NN-index of 1 in TN90, one remained at NN-index 1, two had an NN-index of 2, and two had an NN-index of 3; the NN-index gradually increased with growth. These results, combined with those in Table 11, are shown in Table 12. In the F2 population, 49 individuals had an NN-index of 1 or 2 (no high-nornicotine convertors), and 138 individuals had an NN-index of 3 or higher, which was in good agreement with a 1:3 segregation ratio (P = 0.704, chi-square test). This demonstrated that the inheritance mode of the low-reverting, low-nornicotine trait in Matsukawa (Kanto) is monogenic recessive.

[0065] [Table 12]

[0066] From the above, it was revealed that when the GAGA insertion is homozygous, high-nornicotine convertors do not appear or are extremely unlikely to appear, and that the GAGA insertion can be used as an indicator to select tobacco plants with low revertant and low-nornicotine content.

[0067] (6) Genome analysis of Matsukawa (Kanto) and detection of polymorphisms other than GAGA insertions and application to selection The entire genome sequence of Matsukawa (Kanto) was decoded and compared with the genome sequences of K326, a representative yellow rice variety, and TN90, a representative burley variety. Polymorphisms (SNVs [single nucleotide substitutions] and short indels [insertion / deletion]) found around the GAGA insertion between Matsukawa (Kanto) and yellow rice or / and burley varieties were detected. The genome sequence information of Matsukawa (Kanto) was obtained by DNB-SEQ of DNA extracted from seedling leaves. TM The genome sequence of TN90 was obtained by sequencing using a BGI (BGI) system. The genome sequence of TN90 was obtained from publicly available sequence data (accession no. SRR954964 in the GenBank Sequence Read Archive (SRA)). The genome sequence of K326 was obtained from the publicly available K326 reference genome (ftp: / / ftp.solgenomics.net / genomes / Nicotiana_tabacum / edwards_et_al_2017 / assembly / Nitab-v4.5_genome_Chr_Edwards2017.fasta) and publicly available sequence data (accession no. SRR955769 in the GenBank Sequence Read Archive (SRA)). Markers distinguishable from K326 and TN90 were identified: SNVs 1 to 95 (Table 1) and InDels 1 to 21 (Table 2). The GAGA insertion corresponds to InDel 4.

[0068] Among the markers listed in Tables 1 and 2, we used SNV1 and SNV95, the two most distant in the genome sequence. SNV1 is a single-base substitution in the NLP7 gene, located approximately 192 kb upstream from the GAGA insertion. SNV95 is a single-base substitution in the ZF gene, located approximately 398 kb downstream from the GAGA insertion. DNA markers for detecting these markers were developed (Table 13: forward and reverse primers for PCR, sequencing primers for nucleotide sequence determination). Genotyping analysis was performed using DNA samples from the F2 cross between Matsukawa (Kanto) and Burley 21 (No. 3-10). The GAGA insertion type and NN-index results were compared. PCR was performed using a KOD One (Toyobo Co., Ltd.) kit, with one cycle at 94°C for 2 minutes, followed by 40 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 5 seconds, followed by one cycle at 68°C for 5 minutes. The nucleotide sequences of the PCR products were determined by Sanger sequencing and analyzed using sequence assembly software ATGC (GENETYX). The results are shown in Tables 14 and 15.

[0069] [Table 13]

[0070] As shown in Table 14, of the 48 individuals homozygous for the GAGA insertion (InDel4) for which SNV1 data was available, all 48 individuals had the Matsukawa (Kanto) genotype homozygous for SNV1. All of these had an NN index of 1 or 2. However, one individual was found to have the Matsukawa (Kanto) genotype homozygous for SNV1, but was heterozygous for the GAGA insertion (InDel4), resulting in an NN index of 4. From the above, it was revealed that by selecting individuals with the Matsukawa (Kanto) genotype using the SNV1 marker as an indicator, a low-nornicotine type can be selected with a probability of 48 / 49 = 98%. [Table 14]

[0071] Furthermore, as shown in Table 15, of the 48 individuals with homozygous GAGA insertion (InDel4) and SNV95 data available, 47 had the Matsukawa (Kanto) genotype homozygous for SNV95, and one had the Matsukawa (Kanto) genotype heterozygous for SNV95. All of these individuals had NN-indexes of 1 or 2. On the other hand, one individual had the Matsukawa (Kanto) genotype homozygous for SNV95, but was heterozygous for the GAGA insertion (InDel4), resulting in an NN-index of 4. These results demonstrate that selecting individuals with the Matsukawa (Kanto) genotype using the SNV95 marker as an indicator yields a 47 / 48 = 98% probability of selecting a low-nornicotine genotype. The probability of overlooking an individual with an NN-index of 1 was 1 / 48 = 2%.

[0072] [Table 15]

[0073] From the above, it was demonstrated that both the SNV1 and SNV95 markers are markers that can be used to select low-reverting tobacco plants with low nornicotine content with a high probability.

[0074] (7) Analysis of nornicotine ratio in the progeny (BC5F2) of Matsukawa (Kanto) and TN90 Backcrossing was performed using Matsukawa (Kanto) as the primary parent and TN90 as the recurrent parent. TN90 was crossed with pollen from Matsukawa (Kanto) to obtain F1. Theoretically, this cross would produce a 1:1 segregation ratio of individuals heterozygous for both the GAGA insertion mutation and the single-base substitution in NLP7 from Matsukawa (Kanto), and individuals without either mutation. Heterozygous individuals were selected for the GAGA insertion mutation (Indel4) and the single-base substitution in NLP7 (SNV1). The presence or absence of the GAGA insertion was confirmed by PCR using DNA extracted from seedling leaves as a template, as described above, with the same primers (Forward primer 1 and Reverse primer 1) as Target PCR1 in Table 8. The nucleotide sequence of the amplified product was determined using the sequencing primers near the GAGA insertion in Table 10. The single-base substitution in NLP7 was determined by PCR using the same NLP7 forward and reverse primers listed in Table 13. The nucleotide sequence of the amplified product was determined using the sequencing primers for the NLP7 single-base substitution in Table 13. The selected individuals were further crossed with TN90, and the process was repeated five times. The BC5F1 generation, which was crossed with TN90 as the pollen parent, was selfed, resulting in the BC5F2 generation, which was used in field trials. Among the BC5F2 generation, individuals homozygous for both the GAGA insertion mutation and the NLP7 single-base substitution, and individuals null for both the GAGA insertion mutation and the NLP7 single-base substitution (i.e., possessing neither mutation), were selected as seedlings and then transplanted into the field. All individuals homozygous for the GAGA insertion mutation were also homozygous for the NLP7 single-base substitution, and all individuals null for the GAGA insertion mutation were also null for the NLP7 single-base substitution. Plants were grown in a field and stemmed at the flowering stage. 50 days after stemming, the top three leaves were sampled from each individual plant and allowed to dry naturally for 37 days. The dried samples were crushed and analyzed for nicotine and nornicotine using GC-MS. The ratio of nornicotine content to the total nicotine and nornicotine content was shown as the nornicotine ratio. The results are shown in Figure 1. For convenience, individual numbers are assigned in order of lowest nornicotine ratio.The incidence of individuals with a nornicotine ratio of 5% or higher was 7 out of 174 individuals (4.0%) in the GAGA insertion homozygous line and 44 out of 115 individuals (38.3%) in the GAGA insertion null line. The incidence of individuals with a nornicotine ratio of 10% or higher was 1 out of 174 individuals (0.6%) in the GAGA insertion homozygous line and 14 out of 115 individuals (12.2%) in the GAGA insertion null line. These results demonstrate that low-reverting, low-nornicotine individuals can be selected in progeny lines using the GAGA insertion mutation (Indel4) or single-nucleotide substitution (SNV1) in NLP7 derived from Matsukawa (Kanto). [Industrial Applicability]

[0075] The present invention can be used to create varieties or lines of Nicotiana tabacum.

Claims

1. A method for selecting a tobacco plant with a low revertant low nornicotine content, comprising the step of selecting individuals from a breeding progeny line obtained by crossing a Nicotiana tabacum variety, Matsukawa (Kanto), with another Nicotiana tabacum variety or a mutant thereof, which have, as homozygotes, one or more markers of Matsukawa (Kanto), which are selected from the table below and which distinguish the plant from the other variety or its mutant: Here, the markers indicated by SNV are markers based on single base substitutions present at locations corresponding to the positions indicated on chromosome Nt09 of the reference genome sequence of Nicotiana tabacum K326, and "base" indicates the base in Matsukawa (Kanto). The markers indicated by InDel are markers based on insertions or deletions present at locations corresponding to the positions indicated on Nt09 of the reference genome sequence, and "insertion / deletion" indicates the presence or absence of an insertion or deletion in Matsukawa (Kanto). Table 1 Table 2

2. 2. The selection method according to claim 1, wherein the different variety is a burley variety or a flue-cured variety.

3. The selection method according to claim 1 or 2, wherein the one or more markers include at least one of InDel4, SNV1, and SNV95.

4. The selection method according to claim 1 or 2, wherein the one or more markers include at least InDel4.

5. A first step is to cross a Nicotiana tabacum variety, Matsukawa (Kanto), with another Nicotiana tabacum variety or a mutant thereof to obtain a first generation hybrid plant; a second step of repeatedly backcrossing the first generation hybrid plant body with a Nicotiana tabacum other than Matsukawa (Kanto) as a backcross parent, wherein after each backcrossing, individuals having as heterozygotes one or more markers of Matsukawa (Kanto) that are selected from the table below and that distinguish them from the other cultivar or mutant and the backcross parent are selected and used in the next backcrossing; A method for producing a tobacco plant line with a low revertant low nornicotine content, comprising a third step of self-pollinating the individuals obtained as a result of the second step to obtain breeding progeny, and then carrying out the selection method of claim 1 to select individuals having the one or more markers as homozygotes: Here, the markers indicated by SNV are markers based on single base substitutions present at locations corresponding to the positions indicated on chromosome Nt09 of the reference genome sequence of Nicotiana tabacum K326, and "base" indicates the base in Matsukawa (Kanto). The markers indicated by InDel are markers based on insertions or deletions present at locations corresponding to the positions indicated on Nt09 of the reference genome sequence, and "insertion / deletion" indicates the presence or absence of an insertion or deletion in Matsukawa (Kanto). Table 3 Table 4

6. A tobacco plant with a low revertant, low nornicotine content, obtained by the production method of claim 5.

7. It has an endogenous CYP82E4 gene encoding the amino acid sequence shown in SEQ ID NO: 2, Low-reverting, low-nornicotine tobacco plants having homozygous one or more markers selected from the table below (excluding Matsukawa (Kanto), a variety of Nicotiana tabacum): Here, the markers indicated by SNV are markers based on single base substitutions present at locations corresponding to the positions indicated on chromosome Nt09 of the reference genome sequence of Nicotiana tabacum K326, and "base" indicates the base in Matsukawa (Kanto). The markers indicated by InDel are markers based on insertions or deletions present at locations corresponding to the positions indicated on Nt09 of the reference genome sequence, and "insertion / deletion" indicates the presence or absence of an insertion or deletion in Matsukawa (Kanto). Table 5 Table 6

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