Molecular markers for screening tobacco plants resistant to spotted wilt without linkage drag and their application

Abstract

The present invention relates to the field of tobacco breeding, and more particularly to molecular markers for screening for spotted wilt-resistant tobacco plants free of linkage drag and their applications. A method for screening spotted wilt-resistant tobacco plants or germplasm with reduced linkage drag is provided, comprising: (a) screening for spotted wilt-resistant tobacco plants or germplasm and isolating nucleic acid thereof; (b) detecting a first linkage drag locus marker and / or a second linkage drag locus marker in the isolated nucleic acid; the first linkage drag locus marker comprising the NaChr4_2M marker shown in SEQ ID No. 10 and / or the NaChr4_8M marker shown in SEQ ID No. 11; the second linkage drag locus marker comprising the NaChr3_62.6M marker shown in SEQ ID No. 7 and / or the NaChr3_64.6M marker shown in SEQ ID No. 8; and (c) selecting a spotted wilt-resistant tobacco plant or germplasm that does not contain the first linkage drag locus marker and / or the second linkage drag locus marker. This method can be used to obtain spotted wilt-resistant tobacco plants free of linkage drag, providing superior varieties with high spotted wilt resistance for tobacco leaf production.

Description

Technical Field

[0001] The present invention relates to the field of tobacco breeding, and in particular to a molecular marker for screening tobacco plants resistant to spotted wilt without linkage drag and an application thereof. Background Art

[0002] Tobacco spotted wilt disease (TSWD) is a serious disease caused by viruses of the genus Orthotospoviruses. TSWD causes stunted tobacco plants, wrinkled leaves, and dense, necrotic rings that often merge into larger spots, forming irregular necrotic areas. The disease rapidly spreads from the site of infection to the top of the tobacco plant, causing terminal buds to wilt and droop, ultimately leading to necrosis of the entire plant, rendering it economically worthless. Orthotospoviruses belong to the family Tomato Spotted Wilt Viridae of the order Bunyavirales, and Tomato Spotted Wilt Virus (TSWV) is the representative species of this genus. Orthotospoviruses, with their wide host range and the significant economic losses they cause, have become a significant threat to agricultural production. They are recognized as one of the most destructive plant viruses and rank second on the list of the world's most important plant viruses. Breeding varieties resistant to orthotospoviruses is the most economical and effective means of prevention and control, fundamentally addressing the need for green agricultural pest control. However, there are currently no flue-cured tobacco varieties resistant to TSWD in cultivated tobacco (Nicotiana tabacum L.). All existing cultivated tobacco varieties can be infected by TSWV, posing a potential threat to the prevalence and outbreak of TSWV.

[0003] Previous studies have shown that wild relatives contain many excellent alleles for variety improvement. Discovering and effectively utilizing these excellent alleles in wild relatives, such as disease-resistant genes, will help broaden the disease resistance of cultivated varieties and become one of the most important means of improving variety resistance. Previous studies have shown that a wild tobacco in the Nicotiana genus, winged tobacco (Nicotiana alata), has good resistance to TSWV and is the only available source of resistance to tobacco spotted wilt to date. Winged tobacco (N.alata) inoculated with TSWV only showed allergic necrosis symptoms on the inoculated leaves, and the presence of the virus could not be detected in the systemic leaves. Using the ear-shaped tobacco Notophora as a bridge parent, Gajos et al. successfully located the TSWV resistance site (the resistance site was named RTSW site, where RTSW is R esistance to TSWV) was transferred from wild tobacco N. alata to cultivated tobacco (N. tabacum L.), resulting in the breeding material "Polalta" containing a long RTSW introduced segment.

[0004] However, wild relatives often harbor many genes detrimental to agronomic traits. If these genes are closely linked to the target gene, linkage drag can occur, complicating the utilization of superior genes. Polalta, a tobacco variety resistant to spotted wilt, and other cultivated tobacco lines and varieties containing the long RTSW introgression fragment exhibit significant linkage drag, manifested primarily by varying degrees of stunted growth, leaf deformities such as thickened and irregularly twisted veins, thickened leaves and veins, and narrowed leaves, dwarfing, and varying degrees of reduced fertility, including decreased fruit set, shrunken fruit, and reduced seed count per fruit. Genetic relationships suggest that these linkage drags may originate from linkage drag genomic components within the long RTSW introgress fragment that are closely linked or co-segregate with the RTSW locus, but the specific genetic relationships remain unclear. Global tobacco production urgently needs high-quality varieties with high resistance to spotted wilt disease. However, due to the existence of linkage drag, even though the RTSW locus has been transferred into cultivated tobacco (Nicotiana tabacum L.) for more than 40 years, there has been no variety with high resistance to TSWD for commercial cultivation. Summary of the Invention

[0005] To solve the above problems, the present invention provides molecular markers for identifying or screening for spotted wilt-resistant cultivated tobacco (N. tabacum L.) plants (including cultivated tobacco lines and varieties) without linkage drag, as well as a breeding method for spotted wilt-resistant tobacco plants without linkage drag, and uses the molecular markers and methods to obtain spotted wilt-resistant tobacco plants without linkage drag.

[0006] The present invention discloses two linked drag loci that cause leaf and plant developmental abnormalities in spotted wilt-resistant cultivated tobacco (including cultivated tobacco lines and varieties). The first linked drag loci (DEF1) originates from the end of chromosome 4 of Nicotiana alata (N. alata) and can be detected using the NaChr4_2M marker shown in SEQ ID No. 10 and / or the NaChr4_8M marker shown in SEQ ID No. 11. The second linked drag loci (DEF2) originates from chromosome 3 of Nicotiana alata (N. alata) and can be detected using the NaChr3_62.6M marker shown in SEQ ID No. 7 and / or the NaChr3_64.6M marker shown in SEQ ID No. 8. To obtain plants with linkage drag removed in spotted wilt-resistant cultivated tobacco (including cultivated tobacco lines and varieties), the DEF1 and DEF2 loci need to be removed separately or simultaneously in individual tobacco plants containing the RTSW locus.

[0007] The present invention provides a method for detecting linkage drag loci DEF1 and DEF2. Specifically, the molecular markers NaChr4_2M (amplification primers NaChr4_2MF / NaChr4_2MR) and NaChr4_8M (amplification primers NaChr4_8MF / NaChr4_8MR) developed herein can be used to detect the DEF1 locus in a population of backcross or selfed individual plants. If both primer pairs test negative, or if the polymorphic marker and the recurrent parent (excluding the RTSW introduced fragment) are consistent within a genetic distance of approximately 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM from the two markers, the DEF1 locus has been removed from the individual plant. Furthermore, the molecular markers NaChr3_62.6M (its amplification primers are NaChr3_62.6MF / NaChr3_62.6MR) and NaChr3_64.6M (its amplification primers are NaChr3_64.6MF / NaChr3_64.6MR) developed by the present invention can be used to detect the DEF2 site. If the detection results of both pairs of primers are negative, or the polymorphic marker and the recurrent parent (excluding the RTSW introduced fragment) are consistent within a genetic distance of approximately 20cM, 10cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.5cM or less than 0.5cM, it indicates that the DEF2 site in the single plant has been removed.

[0008] The present invention provides a method for screening tobacco plants or germplasm for resistance to spotted wilt with reduced linkage drag, the method comprising:

[0009] (a) screening tobacco plants or germplasm for resistance to spotted wilt and isolating nucleic acid thereof;

[0010] (b) detecting a first-linked drag site marker and / or a second-linked drag site marker in the isolated nucleic acid; the first-linked drag site marker includes the NaChr4_2M marker shown in SEQ ID No.10 and / or the NaChr4_8M marker shown in SEQ ID No.11; the second-linked drag site marker includes the NaChr3_62.6M marker shown in SEQ ID No.7 and / or the NaChr3_64.6M marker shown in SEQ ID No.8;

[0011] (c) selecting tobacco plants or germplasm that are resistant to spotted wilt that do not comprise the first linked drag locus marker and / or the second linked drag locus marker.

[0012] In some embodiments of the present invention, the NaChr4_2MF / NaChr4_2MR primer pair and the NaChr4_8MF / NaChr4_8MR primer pair developed herein can be used to detect molecular markers NaChr4_2M and NaChr4_8M in tobacco backcross or selfed populations containing the RTSW locus to screen for tobacco plants or germplasm with reduced linkage drag and resistance to spotted wilt. If both primer pairs test negative, or if the polymorphic marker and the recurrent parent are consistent within a genetic distance of approximately 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM, the DEF1 locus has been removed from the tobacco plant. Furthermore, the NaChr3_62.6MF / NaChr3_62.6MR primer pair and NaChr3_64.6MF / NaChr3_64.6MR primer pair developed in the present invention can be used to detect the molecular markers NaChr3_62.6M and NaChr3_64.6M to screen tobacco plants or germplasm with reduced linkage drag and resistance to spotted wilt. If the test results of both pairs of primers are negative, or the polymorphic marker and the recurrent parent are consistent within a genetic distance of approximately 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM or less than 0.5 cM from the two markers, it indicates that the DEF2 site in the tobacco plant has been removed.

[0013] In some embodiments of the present invention, the method for screening tobacco plants or germplasm that are resistant to spotted wilt disease includes: isolating nucleic acid from the tobacco plant or germplasm; detecting a spotted wilt disease resistance marker in the isolated nucleic acid, wherein the spotted wilt disease resistance marker includes the NaChr3_59M marker shown in SEQ ID No. 5; and selecting tobacco plants or germplasm containing the spotted wilt disease resistance marker.

[0014] In some embodiments of the present invention, the tobacco plant or germplasm resistant to spotted wilt is obtained through hybrid breeding methods, mutagenesis breeding methods, genome editing breeding methods and / or transgenic breeding methods.

[0015] In some embodiments of the present invention, the second linkage drag site marker also includes the NaChr3_44.2M marker shown in SEQ ID No.1, the NaChr3_54M marker shown in SEQ ID No.2, the NaChr3_57M marker shown in SEQ ID No.3 and / or the NaChr3_58M marker shown in SEQ ID No.4.

[0016] In some embodiments of the invention, the detecting comprises polymerase chain reaction or nucleic acid sequencing.

[0017] In some embodiments of the present invention, a method for selecting a tobacco plant or germplasm for resistance to spotted wilt with reduced linkage drag comprises:

[0018] (a) hybridizing tobacco plants homozygous for the RTSW introduced segment with tobacco plants homozygous for the genotype rtsw (not containing the RTSW introduced segment) to obtain F1 tobacco plants with the genotype RTSW / rtsw, and then hybridizing the F1 tobacco plants with the genotype rtsw to obtain a population of tobacco plants or germplasm for screening for spotted wilt resistance with reduced linkage drag (BC1F1);

[0019] (b) isolating nucleic acid from BC1F1 tobacco plants, and performing PCR detection using a NaChr3_44.2M primer pair, a NaChr3_54M primer pair, a NaChr3_57M primer pair, a NaChr3_58M primer pair, and a NaChr3_59M primer pair, respectively; screening tobacco plants whose test results with the NaChr3_59M primer pair are positive and whose test results with the NaChr3_44.2M primer pair, the NaChr3_54M primer pair, the NaChr3_57M primer pair, and the NaChr3_58M primer pair are all negative;

[0020] (c) performing spotted wilt resistance testing on the tobacco plants screened in step (b);

[0021] (d) hybridizing the tobacco plants that tested positive for spotted wilt resistance in step (c) with tobacco plants that are homozygous for the rtsw genotype to obtain a population material BC2F1;

[0022] (e) Observe the phenotype of the BC2F1 population at the seedling stage and remove any abnormally developed or deformed plants; seedling stage deformities are mainly manifested as leaf developmental abnormalities, including thickened veins, irregular and twisted veins, thickened leaves and veins, and narrowed leaves;

[0023] (f) performing a spotted wilt resistance test on the phenotypically normal individual plants obtained in step (e) or performing a PCR test using the NaChr3_59M primer pair after extracting genomic DNA, and screening tobacco plants with positive spotted wilt resistance test results or positive NaChr3_59M primer pair test results;

[0024] (g) Isolating the nucleic acids of the tobacco plants obtained by screening in step (f), and performing genotyping detection using a NaChr4_2M primer pair, a NaChr4_8M primer pair, a NaChr3_59M primer pair, and a NaChr3_64.6M primer pair, respectively; selecting tobacco plants whose test results for the NaChr3_59M primer pair are positive and whose test results for the NaChr4_2M primer pair, the NaChr4_8M primer pair, and the NaChr3_64.6M primer pair are all negative.

[0025] Among them, the population materials include population materials BC1F1, BC2F1, BC3F1, BC4F1, BC5F1, BC5F2, BC5F3, BC6F1, BC6F2...BCmFn materials obtained by hybridization, backcrossing and self-pollination, where m represents the number of backcrossing generations, and n represents the number of self-pollination generations after backcrossing.

[0026] The present invention also provides a method for cultivating tobacco plants or germplasm with reduced linkage drag and resistance to spotted wilt, the method comprising:

[0027] (a) hybridizing a first tobacco plant or its germplasm with a second tobacco plant or its germplasm to produce progeny tobacco plants or germplasms thereof, wherein the first tobacco plant or its germplasm comprises a spotted wilt resistance marker and does not comprise a first linkage drag site marker and / or a second linkage drag site marker; the spotted wilt resistance marker comprises the NaChr3_59M marker shown in SEQ ID No.5; the first linkage drag site marker comprises the NaChr4_2M marker shown in SEQ ID No.10 and / or the NaChr4_8M marker shown in SEQ ID No.11; the second linkage drag site marker comprises the NaChr3_62.6M marker shown in SEQ ID No.7 and / or the NaChr3_64.6M marker shown in SEQ ID No.8;

[0028] (b) isolating nucleic acid from the progeny tobacco plant or germplasm;

[0029] (c) detecting the spotted wilt resistance marker, the first linked drag locus marker, and the second linked drag locus marker in the isolated nucleic acid, thereby producing a progeny tobacco plant or germplasm that comprises the spotted wilt resistance marker and does not comprise the first linked drag locus marker and / or the second linked drag locus marker.

[0030] In some embodiments of the present invention, the first tobacco plant or germplasm thereof is obtained by a hybrid breeding method, a mutagenesis breeding method, a genome editing breeding method and / or a transgenic breeding method.

[0031] In some embodiments of the present invention, the second linkage drag site marker further includes the NaChr3_44.2M marker shown in SEQ ID No.1, the NaChr3_54M marker shown in SEQ ID No.2, the NaChr3_57M marker shown in SEQ ID No.3 and / or the NaChr3_58M marker shown in SEQ ID No.4.

[0032] In some embodiments of the invention, the detecting comprises polymerase chain reaction or nucleic acid sequencing.

[0033] In some embodiments of the present invention, the first tobacco plant or germplasm thereof and the second tobacco plant or germplasm thereof are selected from Burley type, dark type, flue-cured type, Maryland type, Oriental type or cigar type.

[0034] The present invention also provides a molecular marker set for screening tobacco plants or germplasm with reduced linkage drag and resistance to spotted wilt, which includes a first linkage drag site marker and / or a second linkage drag site marker; the first linkage drag site marker includes the NaChr4_2M marker shown in SEQ ID No.10 and / or the NaChr4_8M marker shown in SEQ ID No.11; the second linkage drag site marker includes the NaChr3_62.6M marker shown in SEQ ID No.7 and / or the NaChr3_64.6M marker shown in SEQ ID No.8.

[0035] In some embodiments of the present invention, the molecular marker set further includes a spotted wilt resistance marker, the nucleotide sequence of which is shown in SEQ ID No.5.

[0036] The present invention also provides a primer set for screening tobacco plants or germplasm with reduced linkage drag and resistance to spotted wilt, comprising: primers for amplifying the NaChr4_2M marker shown in SEQ ID No. 30 and SEQ ID No. 31; primers for amplifying the NaChr4_8M marker shown in SEQ ID No. 32 and SEQ ID No. 33; primers for amplifying the NaChr3_62.6M marker shown in SEQ ID No. 24 and SEQ ID No. 25; and / or primers for amplifying the NaChr3_64.6M marker shown in SEQ ID No. 26 and SEQ ID No. 27.

[0037] In some embodiments of the present invention, the primer set further includes primers for amplifying the NaChr3_59M marker shown in SEQ ID NO.20 and SEQ ID NO.21.

[0038] The present invention also provides a method for screening tobacco plants or germplasms resistant to spotted wilt, the method comprising:

[0039] (a) isolating nucleic acids from tobacco plants or germplasm;

[0040] (b) detecting at least one spotted wilt resistance marker in the isolated nucleic acid; the spotted wilt resistance marker comprises the NaChr3_59M marker shown in SEQ ID No.5;

[0041] (c) selecting tobacco plants or germplasm comprising at least one spotted wilt resistance marker.

[0042] In some embodiments of the present invention, the NaChr3_59M marker tightly linked to the RTSW locus is detected in the isolated nucleic acid; if the amplification product of the NaChr3_59MF / NaChr3_59MR primer pair is positive, or if the polymorphic marker within a genetic distance of approximately 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM from the NaChr3_59M marker expresses the same behavior as the donor parent Polalta, then the tobacco plant has the spotted wilt resistance trait.

[0043] In some embodiments of the invention, the detecting comprises polymerase chain reaction or nucleic acid sequencing.

[0044] In some embodiments of the present invention, the NaChr3_59M-labeled amplification primers are shown as SEQ ID NO.20 and SEQ ID NO.21.

[0045] The present invention also provides a molecular marker for screening tobacco plants or germplasms resistant to spotted wilt, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0046] The present invention also provides molecular marker primers for screening tobacco plants or germplasms resistant to spotted wilt, the nucleotide sequences of which are shown in SEQ ID NO.20 and SEQ ID NO.21.

[0047] The present invention also provides a method for introducing the spotted wilt resistance trait into a tobacco variety, the method comprising: (a) hybridizing a tobacco donor parent (e.g., Polalta) comprising the spotted wilt resistance trait with a tobacco recurrent parent that does not have the spotted wilt resistance trait to produce one or more offspring tobacco plants; (b) genotyping the one or more offspring tobacco plants to detect a polymorphic marker NaChr3_59M linked to the spotted wilt resistance trait; if the NaChr3_59MF / NaChr3_59MR primer pair amplification product is positive, or if the polymorphic marker and donor parent Polalta are within a genetic distance of about 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM, or less than 0.5 cM, the tobacco plant is characterized by the presence of the spotted wilt resistance trait; and (c) selecting an offspring tobacco plant comprising the spotted wilt resistance trait. Preferably, these methods further comprise backcrossing the offspring tobacco plant selected with the tobacco recurrent parent. These methods further comprise: (d) hybridizing the selected offspring plant with itself or with the tobacco recurrent parent to produce one or more further offspring tobacco plants; and (e) selecting further offspring tobacco plants comprising the spotted wilt resistance trait. Preferably, step (e) comprises marker-assisted selection. Generally, the tobacco recurrent parent is an elite variety. Generally, the genotyping step of these methods involves one or more molecular marker assays. Generally, the polymorphic marker used in the method comprises a polymorphism selected from the group consisting of single nucleotide polymorphisms (SNPs), insertions or deletions (Indels) in the dna sequence, simple sequence repeats (SSRs) of the dna sequence, restriction fragment length polymorphisms (RFLPs) and tag SNPs.

[0048] The present invention also provides tobacco plants and / or germplasm identified, produced or selected by the methods of the present invention, as well as any progeny or seeds derived from tobacco plants or germplasm identified, produced or selected by these methods.

[0049] Those skilled in the art will appreciate that methods for obtaining spotted wilt-resistant tobacco plants with reduced linkage drag, spotted wilt-resistant tobacco plants with DEF1 and / or DEF2 sites removed, and tobacco plants containing a short RTSW-introduced fragment include, but are not limited to, hybrid breeding, chemical or physical mutagenesis, genome editing methods using biotechnology, and de novo gene synthesis. Chemical mutagenesis methods include treatment with mutagens such as sodium azide, ethidium bromide, and ethyl methanesulfonate. Physical mutagenesis methods include treatment with X-rays, gamma rays, fast neutrons, heavy ions, and ultraviolet radiation. Genome editing methods using biotechnology include gene editing technologies such as CRISPR / Cas9, zinc finger endonucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Methods for removing the DEF1 and / or DEF2 sites on the RTSW-introduced fragment or mutating the DEF1 and / or DEF2 sites on the RTSW-introduced fragment to render them inoperable also fall within the scope of the present invention.

[0050] Those skilled in the art will appreciate that the tobacco plant used in the method described herein can be burley type, dark type, flue-cured tobacco type, Maryland type, oriental type or cigar.The tobacco plant used in the method described herein is generally from common tobacco, and can be from multiple common tobacco varieties. The variety may be, but is not limited to, K326, Yunyan 87, Yunyan 97, Yunyan 85, Yunyan 116, Yunyan 121, NC89, Zhongyan 100, Honghua Dajinyuan, Cuibi No. 1, BU 64, CC 101, CC200, CC 13, CC 27, CC 33, CC 35, CC 37, CC65, CC 67, CC 301, CC 400, CC 500, CC 600, CC700, CC 800, CC 900, CC 1063, Coker 176, Coker 319, Coker 371Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 338, GL350, GL 395, GL 600, GL 737, GL 939, GL 973, GF157, GF 318, RJR 901, HB 04P, K 149, K326, K 346, K 358, K394, K 399, K 730, NC 196, NC37NF, NC 471, NC 55, NC 92, NC2326, NC95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH2254, PVH 2275, VA116, VA119, KDH 959, KT200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY171, KY 907, KY907LC, KTY14 x L8 LC, Little Crittenden, McNair 373, McNair 944, msKY14xL8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC4, NC 5, NC 6, NC7, NC 606, NC 71, NC72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, 'Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168. Speight172, Speight179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359.

[0051] It is well known to those skilled in the art that the primer sequences shown herein can be increased by 1 to 30 bases or any chemical modification at their 5' or 3' ends, respectively. The type of bases increased and the chemical modification can be determined based on the base type of the matching region on tobacco genomic DNA and according to the base pairing principle. The resulting primer pairs can produce amplification products substantially identical to the primer sequences shown herein (the DNA sequences between the upstream and downstream primers are identical). Therefore, primer pairs that have increased by 1 to 30 bases or any chemical modification at their 5' or 3' ends, respectively, and can amplify substantially identical DNA fragments, are included in the primer pairs of the present invention. In a specific embodiment of the present invention, the primer pairs of the present invention are preferably the sequences shown in SEQ ID No. 12-33.

[0052] Those skilled in the art will appreciate that examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detecting polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for detecting RNA transcripts, enzyme assays for detecting ribozyme activity of enzymes or polypeptides and polynucleotides, and protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays 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 polynucleotides. Methods for implementing all of the cited techniques are known.

[0053] definition

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although it is believed that the following terms may be well understood by one of ordinary skill in the art, the following definitions are provided to facilitate understanding of the present disclosure.

[0055] The term "RTSW gene" refers to a gene in the genome of Nicotiana tabacum (N. alata) that confers spotted wilt resistance to Nicotiana tabacum (N. alata).

[0056] The terms "RTSW locus," "RTSW resistance locus," "TSWV resistance locus," "spotted wilt resistance locus," or "spotted wilt resistance gene locus" refer to a DNA segment in the genome of Nicotiana alata that contains the RTSW gene, which confers resistance to tobacco spotted wilt in either the heterozygous or homozygous state.

[0057] The term "RTSW introduced fragment" or "resistance introduced fragment" refers to a DNA fragment of Nicotiana alata containing the RTSW gene. Generally, the fragment containing the "RTSW introduced fragment" or "resistance introduced fragment" confers tobacco plants with resistance to tobacco spotted wilt.

[0058] The term "short RTSW introduced fragment" or "short resistance introduced fragment" refers to a DNA fragment in which the non-target gene component linked to the RTSW gene (referred to as the linkage drag gene component) on the long RTSW introduced fragment is partially or completely deleted, while retaining the complete RTSW gene function.

[0059] The term "introgression" or "introgress" refers to the transfer of a desired allele of a genetic locus from one genetic background to another.

[0060] The term "linkage drag" refers to the linkage or co-segregation between favorable genes (target genes) and unfavorable genes (non-target genes) during the breeding process, resulting in the introduction of unfavorable genes along with the favorable genes, often resulting in the new varieties with improved traits being inconsistent with the original target. This type of linkage drag is common during distant hybridization and backcrossing. In this article, linkage drag in spotted wilt-resistant cultivated tobacco (N. tabacum L.) plants (including cultivated tobacco lines and varieties) manifests as varying degrees of developmental delay, short plant stature, leaf deformities including thickened veins, irregular and twisted veins, thickened leaves and veins, and narrowed leaves, as well as varying degrees of reduced fertility, such as decreased fruit set, shrunken fruit, and reduced seed yield per fruit.

[0061] The term "linked drag gene component" refers to a genomic component that contains a drag gene (non-target gene) on a long RTSW-introduced segment but does not contain an RTSW gene (target gene). A "linked drag gene component" can include one or more "linked drag loci." In spotted wilt-resistant cultivated tobacco (N. tabacum L.) plants, the "linked drag gene component" is derived from the N. alata genome, specifically a segment derived from N. alata chromosome 4, which can be detected using molecular markers NaChr4_2M and NaChr4_8M; and a shortened segment derived from N. alata chromosome 3, which can be detected using molecular markers NaChr3_44.2M, NaChr3_54M, NaChr3_57M, NaChr3_58M, NaChr3_62.6M, and NaChr3_64.6M.

[0062] The term "linked marker" means that a marker is "linked" to a trait when it is linked to the trait and when the presence of the marker indicates whether and / or to what extent a desired trait or trait form will occur in the plant / germplasm containing the marker. Similarly, a marker is "linked" to an allele when the presence of the marker indicates whether the allele is present in the plant / germplasm containing the marker. For example, a "marker linked to tobacco spotted wilt resistance" refers to a marker whose presence or absence can be used to predict whether and / or to what extent a tobacco plant will display a TSWD resistance phenotype (e.g., a "linked marker" for TSWD resistance in tobacco plants, such as any of the molecular markers described in Tables 3, 5, and 7).

[0063] The term "spotted wilt resistance marker" refers to a DNA fragment on the RTSW introduced fragment that is closely linked to the RTSW gene (target gene).

[0064] The term "linked drag site marker" refers to a DNA fragment on the RTSW introduced fragment that is tightly linked to a drag gene (non-target gene).

[0065] The term "trait" refers to one or more detectable characteristics of a cell or organism that can be affected by genotype. The phenotype can be observed with the naked eye, or by any other evaluation means known in the art, such as microscopy, biochemical analysis, genetic analysis, specific disease tolerance assays, etc. In some cases, the phenotype is directly controlled by a single gene or genetic locus, such as a "monogenic trait." In other cases, the phenotype is the result of several genes.

[0066] The term "locus" is a chromosomal region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. A locus encompasses one or more polymorphisms in a population; for example, alternative alleles exist in some individuals. The term "allele" refers to an alternative nucleic acid sequence at a specific locus. Alleles can be as small as 1 nucleotide base in length, but are typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome. For example, they can occur on different chromosomes in heterozygous individuals, or between different homozygous or heterozygous individuals in a population.

[0067] The term "chromosomal interval" refers to a continuous linear span of genomic DNA located on a single chromosome. The chromosomal region in the "locus" herein can also be replaced by "chromosomal interval." The term "chromosome" herein is also represented as "Chr."

[0068] The term "centimorgan (cM)" is a unit of measurement for recombination frequency, with one cM being equal to 1% of the probability that the site will separate from the target site. The Kosambi function (Kosambi, The estimation of map distances from recombination values. Annals of Eugenics, 12: 172–75 (1944)) can be used to calculate the genetic distances involved herein from the recombination value. As used herein, "closely linked to ... " or "associated with ... " refers to a marker or locus within about 20 cM, 10 cM, 5 cM, 1 cM, 0.5 cM or less than 0.5 cM of another marker or locus. For example, 20 cM means that the recombination frequency between the marker and the locus is equal to or less than about 20%.

[0069] The term " genotype " refers to that the proterties (phenotype) that is observable and / or detectable and / or shown is by the heredity composition of the individuality (or individual group) at one or more genetic loci.Genotype is defined by one or more alleles of one or more known loci of its parent by individual inheritance." genotype " can be used to refer to the heredity composition of single locus, multiple loci, or more generally, the term " genotype " can be used to refer to the individual inheritance composition of all genes in its genome.Can for example use mark to indirectly characterize genotype and / or directly characterize genotype by nucleic acid sequencing.

[0070] The terms "backcross" and "backcrossed" refer to a method whereby a progeny plant is repeatedly backcrossed to one of its parents. In a backcross protocol, the "donor" parent refers to the parent plant with the desired allele or locus to be introgressed. The "recipient" parent (used one or more times) or the "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on. Specifically in the present invention, the superior parent K326 is the recurrent parent, and the Polalta breeding material is the "donor" parent.

[0071] The result of the plant breeding program using tobacco plant as herein described comprises that the disclosure is, cultivar, kind, offspring, backcross line and hybrid.Term " cultivar " and " kind " refer to can be distinguished by structure or genetic characteristics and / or performance and the plant of one group of similarity in the same species other kinds.Kind is normally, although not always, commercial sale.Although have one or more distinguishing proterties, the further feature of kind is that the overall difference between the individual in this kind is very little.Can be by several generations of self-pollination and selection, or use tissue or cell culture technique to generate " pure line " kind from single parent asexual reproduction.Kind can be derived from another system or kind basically. According to the definition of the International Convention for the Protection of New Varieties of Plants (December 2, 1961, as revised at Geneva on November 10, 1972, October 23, 1978, and March 19, 1991), a variety is "substantially derived" from an initial variety if: a) it is derived primarily from the initial variety, or from a variety derived primarily from the initial variety, while retaining the expression of the essential characteristics resulting from the genotype or combination of genotypes of the initial variety; b) it is clearly distinguishable from the initial variety; and c) except for the differences resulting from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics resulting from the genotype or combination of genotypes of the initial variety. For example, an essentially derived variety can be obtained by selecting natural or induced mutants, somatic variation, variant individuals from plants of the initial variety, backcrossing, or transformation. Unlike a variety, a "line" most commonly refers to a group of plants used non-commercially, such as for plant research. A line typically displays very little overall variation between individuals in one or more traits of interest, although some variation between individuals in other traits may exist.

[0072] The term "selecting" or "selection" refers to the act of picking or choosing desired individuals, typically from a population, based on certain predetermined criteria, in the context of marker-assisted selection or breeding.

[0073] The term "superior strain" or "superior variety" is an agronomically advantageous strain that is produced through many cycles of selection for a particular advantageous agronomic performance. Numerous superior strains are available and are known to those of ordinary skill in the field of tobacco breeding. Similarly, "superior germplasm" or superior varieties of germplasm are agronomically advantageous germplasms, specifically in terms of germination rate, emergence rate, seed vigor, herbicide resistance, insect resistance, disease resistance; high yield; high grade index value; ripening ability; ripening quality; mechanical harvesting performance; ripening tolerance; leaf quality; height, plant maturity (e.g., early maturity, early to mid-maturity, mid-maturity, mid-late maturity, or late maturity); stem size (e.g., small, medium, or large stem); or number of leaves per plant (e.g., few (e.g., 5-10 leaves), medium (e.g., 11-15 leaves), or many (e.g., 16-21) leaves) or any combination thereof.

[0074] The term "hypersensitive reaction" or "HR" refers to a typical disease resistance response in plants that occurs after incompatible plant-pathogen interactions, characterized by rapid cell death. It is a plant disease resistance mechanism accompanied by programmed cell death. Specifically, it manifests as a burst of cellular reactive oxygen species, a rapid response of relevant disease resistance marker genes, and localized cell death.

[0075] The term "avirulence gene" or "avirulence gene NSm" refers to the gene-for-gene hypothesis, which states that for every host resistance gene, the pathogen species has a corresponding avirulence gene. Only when a pathogen carrying an avirulence gene infects a host plant carrying the corresponding resistance gene will it induce plant resistance; otherwise, the plant will become infected and pathogenic. In this invention, the avirulence gene NSm is specifically derived from the NSm protein of TSWV, which is capable of inducing a hypersensitive reaction (HR) in host plants carrying the resistance gene RTSW.

[0076] The HR method induced by avirulent genes can detect tobacco spotted wilt resistance. This method, disclosed in Chinese Patent ZL201710414755.X, uses the NSm gene of tomato spotted wilt virus to identify tobacco resistance. The method comprises the following steps: (a) infiltrating an Agrobacterium suspension carrying the NSm gene into the interveinal space of the tobacco leaves of the intended host plant, specifically: incubating the Agrobacterium containing the NSm gene in LB culture medium for 24 hours at 28°C, collecting the cells by centrifugation, and then diluting the cells with infiltration buffer to an OD value of 0. 600= 0.5; using a sterile syringe without the needle, 9.5-10.5 μL of the bacterial suspension is injected into the interveinal space of the tobacco leaves through the back of the tobacco plants, forming a visible infiltration spot; the inoculated tobacco plants are placed in an environment of 20-28°C and 80% humidity, with alternating continuous light exposure for 16 hours and continuous darkness for 8 hours, for a total of 72 hours. (b) Detecting the allergic reaction of the test host, specifically comprising: using the EHA105 strain containing pK2-35S-NSm + p2300-35S-Sw-5b as a positive control and the EHA105 strain containing pK2-35S-NSs as a negative control on the tobacco host, and observing. If the tobacco host produces an allergic reaction induced by the identification strain containing the avirulent gene NSm expression vector, the tested tobacco plants are confirmed to be tobacco plants with spotted wilt resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The results showed that after five generations of backcrossing using tobacco material Polalta as the male parent and the main susceptible variety K326 (Nicotiana tabacumcv. K326) as the female parent, the phenotype of linkage drag containing the spotted wilt resistance locus was obtained. Figure 1 A shows the leaf deformity phenotype, including thickened veins, irregularly twisted veins, thickened leaves and veins, and narrowed leaves; Figure 1 B and C show the deformed phenotype of the whole plant, which is manifested by stunted growth, twisted stems, and short plants.

[0078] Figure 2 The distribution of SNP sites between the resistant pool (RTSW-pool) and the susceptible pool (rtsw-pool) found by using the delta SNP-index and Euclidean distance (ED) algorithm is shown at 43.7Mb to 64.8Mb (Chr3:43.7-64.8Mb) on chromosome 3 of the N.alata genome.

[0079] Figure 3 The results of the analysis of the malformation-susceptible pool (DEF_rtsw_pool) and the normal-susceptible pool (def_rtsw_pool) using the delta SNP-index and Euclidean distance (ED) algorithms are shown. BSA analysis revealed that the DEF1 locus, which causes malformations, is located within the 1-8.6 Mb range at the end of chromosome 4.

[0080] Figure 4The predicted genetic models of DEF1 and RTSW (DEF2) are shown. The DEF1 locus originates from chromosome 4, 1-8.6Mb, of wild tobacco N.alata. DEF2 and the resistance locus (RTSW locus) are tightly linked and located at the same locus, originating from chromosome 3, 43.7Mb to 64.8Mb, of wild tobacco N.alata. It is speculated that there may be a tightly linked toxic gene (Toxin) near the RTSW (DEF2) locus. The presence of the RTSW (DEF2) locus alone may lead to more severe deformity phenotypes or affect fertility and other factors. The DEF1 locus may be tightly linked to a detoxification gene (Detoxin). Only in the presence of the DEF1 locus can the RTSW (DEF2) locus be stably inherited. The markers used in the present invention have been marked in the figure.

[0081] Figure 5 Shown are partial results from screening using the molecular markers NaChr3_59M and NaChr3_64.6M located at either end of the resistance-introduced segment in strain 46. Simultaneous screening of these markers at this locus, using an optimized molecular marker screening system, yielded 18 strains harboring at least one of these markers.

[0082] Figure 6 Shown are phenotypic observations at the cluster and mature plant stages. A, Five individual plants selected from 160,000 tobacco plants that harbor the RTSW resistance locus and completely eliminate linkage drag. B, Typical spotted wilt-resistant tobacco plants with linkage drag. C, Mature phenotype of individual plant No. 12, whose key agronomic traits, such as plant height, leaf foliation, and flowering time, are indistinguishable from those of the conventional K326 variety.

[0083] Figure 7 The results of virus inoculation testing of the self-pollinated progeny of individual plant No. 12 are shown. Genotyping of the self-pollinated progeny of individual plant No. 12 was performed using the primers NaChr3_59MF / NaChr3_59MR, which amplify the NaChr3_59M molecular marker. Marker-positive (labeled RTSW in the figure) and marker-negative (labeled rtsw in the figure) plants were selected and friction-inoculated with Tomato Spotted Wilt Virus (TSWV). After two weeks of indoor cultivation, TSWV symptoms were observed. The inoculation revealed that plants containing the introduced resistance fragment were completely resistant to the disease, while plants without the introduced resistance fragment developed 100% disease.

[0084] Brief Description of Sequence Listing

[0085] SEQ ID No. 1 is the nucleotide sequence of the NaChr3_44.2M marker;

[0086] SEQ ID No. 2 is the nucleotide sequence of the NaChr3_54M marker;

[0087] SEQ ID No. 3 is the nucleotide sequence of the NaChr3_57M marker;

[0088] SEQ ID No. 4 is the nucleotide sequence of the NaChr3_58M marker;

[0089] SEQ ID No. 5 is the nucleotide sequence of the NaChr3_59M marker;

[0090] SEQ ID No. 6 is the nucleotide sequence of the NaChr3_60M marker;

[0091] SEQ ID No. 7 is the nucleotide sequence of the NaChr3_62.6M marker;

[0092] SEQ ID No. 8 is the nucleotide sequence of the NaChr3_64.6M marker;

[0093] SEQ ID No. 9 is the nucleotide sequence of the NaChr3_65.7M marker;

[0094] SEQ ID No. 10 is the nucleotide sequence of the NaChr4_2M marker;

[0095] SEQ ID No. 11 is the nucleotide sequence of the NaChr4_8M marker;

[0096] SEQ ID No. 12 is the upstream primer used to amplify the NaChr3_44.2M marker;

[0097] SEQ ID No. 13 is the downstream primer used to amplify the NaChr3_44.2M marker;

[0098] SEQ ID No. 14 is the upstream primer used to amplify the NaChr3_54M marker;

[0099] SEQ ID No. 15 is a downstream primer for amplifying the NaChr3_54M marker;

[0100] SEQ ID No. 16 is the upstream primer used to amplify the NaChr3_57M marker;

[0101] SEQ ID No. 17 is a downstream primer for amplifying the NaChr3_57M marker;

[0102] SEQ ID No. 18 is the upstream primer used to amplify the NaChr3_58M marker;

[0103] SEQ ID No. 19 is a downstream primer for amplifying the NaChr3_58M marker;

[0104] SEQ ID No. 20 is the upstream primer used to amplify the NaChr3_59M marker;

[0105] SEQ ID No. 21 is a downstream primer for amplifying the NaChr3_59M marker;

[0106] SEQ ID No. 22 is the upstream primer used to amplify the NaChr3_60M marker;

[0107] SEQ ID No. 23 is a downstream primer for amplifying the NaChr3_60M marker;

[0108] SEQ ID No. 24 is the upstream primer used to amplify the NaChr3_62.6M marker;

[0109] SEQ ID No. 25 is the downstream primer used to amplify the NaChr3_62.6M marker;

[0110] SEQ ID No. 26 is the upstream primer used to amplify the NaChr3_64.6M marker;

[0111] SEQ ID No. 27 is the downstream primer used to amplify the NaChr3_64.6M marker;

[0112] SEQ ID No. 28 is the upstream primer used to amplify the NaChr3_65.7M marker;

[0113] SEQ ID No. 29 is the downstream primer used to amplify the NaChr3_65.7M marker;

[0114] SEQ ID No. 30 is the upstream primer used to amplify the NaChr4_2M marker;

[0115] SEQ ID No. 31 is a downstream primer for amplifying the NaChr4_2M marker;

[0116] SEQ ID No. 32 is the upstream primer used to amplify the NaChr4_8M marker;

[0117] SEQ ID No. 33 is the downstream primer used to amplify the NaChr4_8M marker. DETAILED DESCRIPTION

[0118] The present invention is described in further detail below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are conventional products that can be purchased.

[0119] The tobacco material Polalta is a TSWV-resistant tobacco material containing the RTSW locus (spotted wilt resistance gene locus) and has been described in non-patent literature (Laskowska D, A,2010.TSWV resistance in DH lines of tobacco(Nicotiana tabacum L.)obtained from a hybrid between'Polalta'and The tobacco variety K326 (Nicotiana tabacum cv. K326), a TSWV-susceptible tobacco material lacking the RTSW locus (spotted wilt resistance locus), has been published in the non-patent literature (Edwards et al., 2017, A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. Bmc Genomics 18, 448). Its reference genome sequence is available at https: / / solgenomics.net / organism / Nicotiana_tabacum / genome. BC5F3, BC6F1, and BC6F2 progeny populations obtained by hybridization, backcrossing, and selfing of Polalta with K326 were created and preserved by our research group. Nicotiana alata is a wild tobacco plant resistant to TSWV, which has been disclosed in non-patent literature (Laskowska et al., 2013, Survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus (TSWV). Euphytica 193, 207-19.). The Nicotiana alata plant used in the present invention has accession number PI42334 in the American Tobacco Germplasm Bank. The above tobacco materials are all common tobacco germplasm resources and are publicly available from tobacco germplasm resource conservation institutions or the Yunnan Tobacco Agricultural Science Research Institute.

[0120] The TSWV virus source and Agrobacterium tumefaciens EHA105 carrying the avirulence gene NSm, used for resistance testing, are stored at the Yunnan Tobacco Agricultural Science Research Institute. The preparation method of Agrobacterium tumefaciens EHA105 carrying the avirulence gene NSm is described in Chinese Patent No. ZL201710414755.X, entitled "A Method for Identifying Tobacco Resistance Using the NSm Gene of Tomato Spotted Wilt Virus," which is hereby incorporated by reference in its entirety.

[0121] Reagents: All molecular biology-related reagents, instruments, and consumables were commercially available.

[0122] Example 1. Obtaining a BC6F2 segregating population and defining linkage drag for spotted wilt resistance

[0123] The material Polalta (♂) containing the RTSW spotted wilt resistance locus was used as the male parent and hybridized with the main susceptible variety K326 (♀) (Nicotiana tabacum cv. K326) as the female parent. Five backcross generations were performed with K326 as the recurrent parent, and selection was performed based on the disease resistance trait to obtain the homozygous high-generation tobacco material BC5F3. After multiple generations of backcrossing, the spotted wilt resistance was stable, but the deformed phenotype still existed without any signs of alleviation. The specific manifestations are varying degrees of developmental delay, short plants, leaf deformities including thickened leaf veins, irregular and twisted leaf veins, thickened leaves and leaf veins, narrowed leaves, etc., as well as varying degrees of reduced fertility, such as decreased fruit set rate, shriveled fruits, and decreased seed quantity per fruit ( Figure 1 ). Using the homozygous tobacco high-generation material BC5F3 as the male parent and the main susceptible variety K326 as the recurrent parent, backcrossing was continued to obtain BC6F1, and self-pollination was used to obtain the BC6F2 population. Using the non-toxic gene infiltration identification method established by this project team, 454 individual plants in the BC6F2 population were identified for spotted wilt resistance. The non-toxic gene infiltration identification method is a method for identifying tobacco resistance using the tomato spotted wilt virus NSm gene, which is recorded in the Chinese patent with patent number ZL201710414755.X and invention name "A method for identifying tobacco resistance using the tomato spotted wilt virus NSm gene". The specific operation steps are:

[0124] (1) Agrobacterium EHA105 containing the non-toxic gene NSm expression vector was cultured in LB medium for 24 h at 28°C. The cells were collected by centrifugation and diluted with infiltration buffer (10 mmol / L MgCl2, 10 mmol / L MES, 200 μmol / L acetosyringone) to an OD of 600 =0.5 bacterial suspension;

[0125] (2) Using a sterile syringe with the needle removed, inject 9.5-10.5 μl of the bacterial suspension from the back of the tobacco leaf into the interveinal space, forming a visible infiltration spot. Place the inoculated tobacco plant in an environment of 20-28°C and 80% humidity, alternating between 16 hours of continuous light and 8 hours of continuous darkness, and observe for a total of 72 hours.

[0126] (3) Observation: If the tobacco test host produces a hypersensitive reaction (HR) induced by the identified strain containing the avirulent gene NSm expression vector, it is confirmed that the tobacco test host is a disease-resistant variety relative to the avirulent gene NSm.

[0127] Through NSm-mediated disease resistance identification, 319 out of 454 plants in the BC6F2 population produced HR reactions and were resistant plants (indicated by RTSW), 118 plants had no HR reactions and were susceptible plants (indicated by rtsw), and the other 17 plants were not included in the statistics because they were too small to be suitable for HR detection.

[0128] We further verified the genetic relationship of the spotted wilt resistance loci using the HR results. A chi-square test was performed on the resistance traits of the BC6F2 generation population. Under the premise of an expected ratio of 3:1, χ 2 =0.9344, when df=1, χ 2 0.05 =0.3337, the test result χ 2 The difference was not significant, indicating that the segregation of resistance traits in the BC6F2 generation population conformed to Mendel's law of inheritance. The chi-square test confirmed that the RTSW gene-mediated spotted wilt resistance was a single gene dominant trait (see Table 1 for results).

[0129] Table 1 Segregation ratio of resistance traits in BC6F2 population

[0130]

[0131] At the same time, we conducted detailed observations and statistical analyses of the deformity phenotypes. Based on the observations of the deformity phenotypes, we further verified the genetic relationship of the deformity gene loci. In the statistical process of the deformity phenotypes, we classified all phenotypes that affect the development of plants and leaves to varying degrees as deformity phenotypes, represented by DEF (Deformity). Those with completely normal development and no deformity phenotypes were classified as normal, represented by def. A chi-square test was performed on the resistance traits of the BC6F2 generation population, because the approximate deformity (DEF): normal (def) was 417:37 = 11.27:1, which was much higher than 3:1 and closer to the separation ratio of 15:1. Therefore, under the premise that the expected ratio was 15:1, χ 2 =2.79, when df=1, χ 2 0.05 =0.094, the test result χ 2 >0.094, indicating that the difference was not significant, proving that the deformity traits of the BC6F2 generation population conform to Mendel's law of inheritance and are presumed to be controlled by two genes.

[0132] What is even more interesting is that we also analyzed the subpopulation BC6F2:S, which is composed of all susceptible plants in the BC6F2 population. Among the BC6F2 population, there are 118 susceptible plants, and the ratio of deformed (DEF): normal (def) is 81:37 = 2.18:1, which is closer to the segregation ratio of 3:1. Therefore, under the premise of the expected ratio of 3:1, χ 2=2.54, when df=1, χ 2 0.05 =0.111, test result χ 2 >0.111, indicating that the difference was not significant, proving that the deformity traits in the susceptible subpopulation BC6F2:S in the BC6F2 generation population conform to Mendel's law of single gene inheritance (results are shown in Table 2).

[0133] Table 2 Segregation ratio of deformity phenotypes in the BC6F2 generation

[0134] Generation (group) deformity normal Expectation ratio Chi-square value degrees of freedom (df) Asymptotic significance BC6F2 417 37 15:1 2.7964 1 0.094472 BC6F2:S(susceptible) 81 37 3:1 2.542 1 0.111

[0135] Phenotypic analysis and chi-square test showed that the deformity phenotype in the population was controlled by multiple genes, and there were probably two loci controlling the plant deformity phenotype.

[0136] Example 2. Initial location of the spotted wilt resistance locus and development of molecular markers

[0137] In order to exclude the influence of deformed phenotype on the resistance to spotted wilt loci, we randomly selected 40 deformed plants from 319 resistant plants and 118 susceptible plants to construct a resistance pool (RTSW-pool) and a susceptible pool (rtsw-pool). The construction method was to take 0.1g of leaves from each of the 40 selected disease-resistant plants, a total of 4g, and mix them to construct a resistance pool (RTSW-pool) and send them for DNA resequencing. The same method was used to construct the susceptible pool (rtsw-pool) and send them for sequencing. At the same time, the disease-resistant parent Polalta and the susceptible parent K326 were also sent for sequencing as parents. The sequencing depth was 30X, and 135G data was sequenced for each sample. Sequencing was carried out using the BGI500 sequencing platform of BGI. The resistant pool / susceptible pool data obtained by resequencing were aligned to the chromosome-level N. alata genome. Using BSA analysis and delta SNP-index, a total of 7774 SNP sites with a Delta value greater than 0.5 and a significance greater than 99% between the resistant pool and the susceptible pool were found. Among them, 7732 SNP sites were distributed between 43.7Mb and 64.8Mb on chromosome 3 of the N. alata genome (Chr3: 43.7-64.8Mb). The same results were obtained using the Euclidean distance (ED) algorithm (see Figure 2 ).

[0138] Example 3. Development and validation of molecular markers for resistance to spotted wilt

[0139] Based on the SNPs identified in the BSA analysis of the resistant / susceptible pools, we concluded that the resistance fragment in the spotted wilt-resistant tobacco in the BC6 population originated from a region between 43.7 and 64.8 Mb on chromosome 3 of the N. alata genome, with a size of 21.1 Mb. To further shorten the size of the resistance fragment, we designed molecular markers near the left and right ends of the introduced fragment (Table 3).

[0140] Table 3 Molecular markers of resistance-introduced fragments (1)

[0141]

[0142] To validate the markers, we used Nicotiana tabacum (N. alata), Polalta, a spotted wilt-resistant tobacco variety, K326, a BC6F1 population, and 437 BC6F2 individuals identified as both spotted wilt-resistant and susceptible. Marker validation was performed as follows: Genomic DNA was extracted from leaves of the test tobacco plants using a Plant Genomic DNA Extraction Kit (Tiangen Biochemical, Cat. No. DP360) according to the kit's instructions. For each marker, PCR amplification was performed using genomic DNA from test tobacco leaves as a template using a primer pair consisting of two single-stranded DNAs. The PCR reaction system consisted of 12.5 μL of 2× Premix Ex TaqMix PCR Buffer (Takara, Cat. No. RR003A), 0.5 μL of a 10 μmol / L forward primer, 0.5 μL of a 10 μmol / L reverse primer, and 1 μL of 50 ng / μL template DNA. Sterile double-distilled water was added to bring the total volume to 25 μL. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes; then enter 35 cycles: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; after the cycle, extension at 72°C for 10 minutes; and storage at 4°C. After the reaction, the PCR products were electrophoresed using a ZAG DNA analyzer system (Agilent, model M5320AA). If the expected PCR product is detected, the marker detection result is positive; if the expected PCR product is not detected, the marker detection result is negative. The above marker verification method is applicable to the verification or detection of all markers disclosed herein.

[0143] The results showed that marker NaChr3_65.7M was negative in all tobacco plants, including K326, BC6F1, and BC6F2 populations, except for N.alata and Polalta. Markers NaChr3_44.2M and NaChr3_64.6M were positive in Polalta, N.alata, BC6F1 populations, and 319 individual BC6F2 plants resistant to spotted wilt, but negative in K326 and 118 susceptible BC6F2 plants. These markers are tightly linked to spotted wilt resistance, co-segregating with the spotted wilt-resistant phenotype in all generations. Furthermore, these markers are located at both ends of the resistance-introduced segment, making them suitable for later detection of shortened segments.

[0144] Example 4. Preliminary mapping of linkage drag loci and development of molecular markers

[0145] Using resistance screening and observation of malformation phenotypes, the BC6F2 population was initially divided into three subpopulations: malformation-resistant (genotype DEF_RTSW), malformation-susceptible (genotype DEF_rtsw), and normally susceptible (genotype def_rtsw). No plants with normal resistance (i.e., genotype def_RTSW) were found. Approximately 40 plants were selected from each of the three subpopulations to construct pools: the malformation-resistant pool (DEF_RTSW_pool), the malformation-susceptible pool (DEF_rtsw_pool), and the normally susceptible pool (def_rtsw_pool). These pools were constructed by taking 0.1g of leaves from each of the 40 selected plants, mixing the total 4g, and then pooling them for DNA resequencing. Sequencing depth was 30X, with 135GB of data sequenced for each sample.

[0146] Through genetic analysis of the initial segregating population, we preliminarily determined that the deformity phenotype is determined by two dominant loci. To accurately locate the first deformity locus (DEF1), we first eliminated the interference of the spotted wilt resistance locus and selected the deformity susceptible pool (DEF_rtsw_pool) and the normal susceptible pool (def_rtsw_pool) for analysis. By aligning the resequencing data of the two mixed pools and the parents to the N. alata genome, we performed △SNP-Index and ED analysis ( Figure 3 SNP typing revealed that the malformation site, DEF1, is located 1-8.6 Mb from the end of chromosome 4 in the N. alata genome. Based on the resequencing results, we designed two pairs of markers near the left and right ends of the introduced fragment (Table 4), located at 2 Mb and 8 Mb from chromosome 4 in the N. alata genome.

[0147] Table 4 Molecular markers at both ends of the first malformation site (DEF1) introduced into the fragment

[0148]

[0149] Using these markers and combined with spotted wilt resistance identification, we selected Polalta, N.alata, K326, BC6F1, and 118 BC6F2 tobacco plants susceptible to malformation (DEF_rtsw) and normal disease (def_rtsw) for marker verification. The marker verification method was the same as that in Example 3.

[0150] Results showed that the NaChr4_2M and NaChr4_8M markers were positive in N.alata, Polalta, BC6F1, and 81 BC6F2 tobacco plants susceptible to the malformation (DEF_rtsw), consistent with the malformation phenotype. However, marker detection was negative in K326 and 37 BC6F2 tobacco plants susceptible to the normal disease (def_rtsw), consistent with the normal phenotype. The NaChr4_2M and NaChr4_8M markers are located at either end of the resistance-introduced segment and are fully linked to the DEF1 locus, making them suitable for detection at the DEF1 locus.

[0151] Example 5. Determination of the second malformation site (DEF2)

[0152] To identify the second malformation locus, we screened individual plants with malformation phenotypes using molecular markers (NaChr4_2M and NaChr4_8M) at either end of the introduced fragment of the first malformation locus (DEF1). We then resequenced a pool of 40 randomly selected tobacco plants (DEF2_pool) that did not harbor the DEF1 introduced fragment. The resulting data were then compared with a completely normal pool (def_pool) for BSA analysis. Delta SNP-Index and ED analyses were also performed. The resequencing data from the DEF2_pool and def_pool were aligned with the genome of the N. alata chromosome. We found that the malformation DEF2 locus and the resistance locus (RTSW) are located in the same region, between 43.7 and 64.8 Mb on chromosome 3 of the N. alata genome, indicating that the DEF2 locus and the resistance locus overlap. We used molecular markers at both ends of the resistance introduction fragment to screen 319 deformed plants that did not contain the first deformity site (DEF1) from the BC6F2 generation population. The marker detection results were completely consistent with the deformity phenotype, which indicates that the DEF2 site should be closely linked to the RTSW site and located near the RTSW site.

[0153] Example 6. Screening of individual plants carrying short resistance fragments

[0154] We used the left-end marker primers NaChr3_44.2MF / NaChr3_44.2MR and the right-end marker primers NaChr3_64.6MF / NaChr3_64.6MR to screen a backcross population. The male BC5F2-4Q individual of this backcross population was a heterozygous type (RTSW / rtsw). By hybridizing with the female K326, we obtained a BC6F1 backcross population of BC5F2-4Q individual plants. 1,500 individual plants from this population were selected for marker screening. The screening principle is that individual plants with both end markers negative or both end markers positive are non-exchange individual plants, while individual plants with only one of the left and right end markers positive are exchange individual plants. Amplification using the NaChr3_44.2MF / NaChr3_44.2MR primer pair revealed a 470bp amplification product in the disease-resistant controls N.alata and Polalta, while no amplification product was observed in the susceptible control K326, indicating normal PCR amplification. Similarly, amplification using the NaChr3_64.6MF / NaChr3_64.6MR primer pair revealed a 494bp amplification product in the disease-resistant controls N.alata and Polalta, while no amplification product was observed in the susceptible control K326, indicating normal PCR amplification. DNA was extracted from leaves collected from 1500 plants in a 4Q×K326 BC6F1 backcross population for PCR marker verification, using the same method as in Example 3. Results: Plants No. 46 and No. 364 were screened for the NaChr3_44.2M marker, but positive for the NaChr3_64.6M marker. Plant No. 59 was screened for the NaChr3_44.2M marker, but negative for the NaChr3_64.6M marker. Identification of spotted wilt resistance (using the same method as in Example 1) revealed that plant No. 59 was not resistant to spotted wilt and showed no HR after inoculation with the avirulent gene. However, strains No. 46 and No. 364 exhibited HR after inoculation with the avirulent gene, indicating they were resistant to spotted wilt and were preliminarily determined to have effectively shortened resistance-introduced segments. To clarify the extent of the shortened resistance-introduced segments in strains No. 46 and No. 364, we further enriched the molecular markers (Table 5).

[0155] Table 5 Molecular markers of resistance-introduced fragments (2)

[0156]

[0157]

[0158] Using these molecular markers, we detected the sizes of the resistance-introduced fragments contained in strains 59, 46, and 364. The results are shown in Table 6.

[0159] Table 6 Marker detection results of exchanged individual plants

[0160]

[0161] Note: "59#," "46#," and "364#" in the table represent "individual plant No. 59," "individual plant No. 46," and "individual plant No. 364," respectively. Pos indicates positive, Neg indicates negative, and HR indicates necrotic hypersensitivity reaction. "HR" indicates resistance to spotted wilt, and "no HR" indicates resistance to spotted wilt.

[0162] As can be seen from Table 6, strain 46 has a shorter resistance segment. The left end of the introduced segment of this strain lost markers ranging from 44.2Mb to 58Mb, shortening the segment by more than 14Mb. Through resistance identification, we speculate that the resistance gene locus is located in the 58Mb and 64.8Mb interval of Chr3 of the N.alata genome.

[0163] We tested individual plant 46 using molecular markers (NaChr4_2M and NaChr4_8M) at either end of the introduced fragment containing the first malformation locus (DEF1) and found it to be positive for DEF1. Because individual plant 46 still contained the DEF1 locus, it still exhibited a severe malformation phenotype. However, our results indicated that the DEF1 locus and RTSW (DEF2) were located on different chromosomes and should be able to segregate freely. Therefore, we attempted to identify offspring from individual plant 46 that contained only the RTSW (DEF2) introduced fragment. Screening of a self-segregating F2 population of 248 individual plants from individual plant 46 revealed 60 plants containing only RTSW (DEF2) but not DEF1, all of which displayed a severe malformation phenotype. Furthermore, 34 of these plants failed to flower at all, while the remaining 26 exhibited generally delayed flowering and a decreased number of flowers. Four of these plants produced only one to two flowers each, with a generally decreased fruit set rate and low seed yield.

[0164] Example 7. Genetic relationships of linked drag loci

[0165] Through analysis of the disease resistance locus (RTSW) and the deformity locus (DEF), we have preliminarily established a genetic relationship between the disease resistance locus (RTSW) and the deformity locus (DEF). We found that the deformity phenotype is determined by two loci. One locus (DEF1) is located on chromosome 4, between 1 and 8.6 Mb, in the wild tobacco (N. alata) genome. The other locus (DEF2) is tightly linked to the resistance locus (RTSW) and is located in the same genomic region, originating from chromosome 3, between 43.7 and 64.8 Mb. Furthermore, DEF1 and RTSW (DEF2) cosegregated and were consistently maintained during multiple generations of backcrossing. Investigations of segregating populations of the progeny of individual plant No. 46 suggest that the deformity locus DEF1 is crucial for the stable presence of the RTSW (DEF2) locus. If only the RTSW (DEF2) locus is present, severe deformities and reduced fertility will result. The presence of both DEF1 and RTSW (DEF2) loci results in severe deformed phenotypes in the plants, but no significant difference in fertility from the parental lines, Polalta and K326. Therefore, we propose the following model: the RTSW (DEF2) locus may harbor a toxic gene (Toxin), whose presence alone may lead to more severe deformed phenotypes or affect fertility, while the DEF1 locus may harbor a detoxification gene (Detoxin), and only in the presence of DEF1 can the RTSW (DEF2) locus be stably inherited. Figure 4 ).

[0166] Example 8. Simultaneous removal of two linkage drag loci using a very large population

[0167] According to our hypothetical model, the RTSW (DEF2) locus may harbor a toxic gene, the presence of which alone could lead to more severe malformation phenotypes or affect fertility. The DEF1 locus, however, likely harbors a detoxification gene, and the RTSW (DEF2) locus can only be stably inherited in the presence of DEF1. Therefore, to eliminate the DEF1 locus, the fertility-affecting gene at the RTSW (DEF2) locus must first be eliminated. To obtain completely normal plants, the DEF2 locus must also be eliminated simultaneously. However, the introduced DEF1 and RTSW (DEF2) fragments from the wild tobacco plant N. alata have low sequence homology to those in cultivated tobacco, leading to recombination suppression and a low recombination exchange rate. To simultaneously shorten the introduced DEF1 and RTSW (DEF2) fragments and obtain completely normal resistant plants, selection would require a large number of segregating populations. The malformation phenotype could be the primary screening target during the seedling stage. To conduct phenotypic screening, we seeded a backcross BC7F1 population in the nursery pond, with plant No. 46 as the male parent (RTSW(DEF2) / rtsw(def2), DEF1 / def1 genotype) and K326 as the female parent. We seeded 1,600 floating trays, with approximately 100 plants per tray, for a total of over 160,000 plants for screening. Initially, we screened only for abnormal phenotypes. During the seedling stage, we conducted five rounds of screening, visually inspecting and promptly removing any abnormalities. Through rigorous screening, we ultimately retained 12,000 completely normal seedlings for molecular marker screening.

[0168] After extracting genomic DNA from 12,000 tobacco plants, we performed marker testing. First, we screened using the molecular markers at both ends of the resistance-introduced fragment in plant 46, namely NaChr3_59M and NaChr3_64.6M. We simultaneously screened for this locus using molecular markers at both ends. Using our optimized molecular marker screening system, we obtained nearly 18 plants with at least one of the markers present ( Figure 5 ).

[0169] Secondly, we used the markers of the DEF1 site (NaChr4_2M and NaChr4_8M) to test these 18 strains. The results of all individual strains tested using the primer pairs NaChr4_2MF / NaChr4_2MR and NaChr4_8MF / NaChr4_8MR were negative, indicating that all individual strains had separated the deformity site DEF1.

[0170] Eighteen individual plants identified through initial molecular marker screening were tested for spotted wilt resistance using avirulence gene-mediated HR (using the same method as in Example 1). By transiently expressing the avirulence gene NSm and observing the HR response, the presence of the resistance-introducing fragment was determined. Five of the 18 plants were identified as resistant to spotted wilt when the avirulence gene was expressed. These five plants were numbered: plant 1, plant 4, plant 11, plant 12, and plant 17.

[0171] To determine the size of the resistance-introduced fragments contained in the five individual plants and select the shortest resistance-introduced fragment for future breeding, we performed encrypted molecular marker testing on the five resistant plants identified. Using the genomic sequence, we further developed two new pairs of molecular marker primers based on the resistance-introduced fragments for detection (Table 7).

[0172] Table 7 Molecular markers of resistance-introduced fragments (3)

[0173]

[0174] Through testing, we found that the single plant with the shortest resistance-introduced fragment (single plant No. 12) was only positive for the NaChr3_59M marker, and all other markers were negative (Table 8).

[0175] Table 8 Marker detection results of exchanged individual plants

[0176]

[0177] Note: "1#," "4#," "11#," "12#," and "17#" in the table represent "Single Plant No. 1," "Single Plant No. 4," "Single Plant No. 11," "Single Plant No. 12," and "Single Plant No. 17," respectively. Pos indicates positive, Neg indicates negative, and HR indicates necrotic hypersensitivity reaction. "HR" indicates resistance to spotted wilt, and "no HR" indicates resistance to spotted wilt.

[0178] Example 9. Phenotype and spotted wilt resistance detection of self-pollinated progeny of single plants with linkage drag sites removed

[0179] Whether the phenotype is completely restored to normal is the key to the present invention. Therefore, we conducted phenotypic observations on the five resistant individual plants (plant No. 1, plant No. 4, plant No. 11, plant No. 12, and plant No. 17) screened in Example 8 during the clustering and mature stages. The observation results showed that the phenotypes of the five resistant individual plants during the clustering and mature stages had no visible linkage drag, and the plant type was significantly improved. The main agronomic traits such as plant height, leaves, and flowering period were close to those of the conventional K326 variety ( Figure 6 ).

[0180] In order to detect whether the resistance-introduced fragment still retains complete resistance to TSWV virus, we inoculated the self-pollinated progeny of single plant No. 12 with TSWV virus. The molecular marker detection primers NaChr3_59MF / NaChr3_59MR were used to perform genotyping on the self-pollinated progeny of single plant No. 12. Molecular marker-positive and -negative plants were selected for friction inoculation with TSWV, and the symptoms were observed after two weeks of indoor cultivation. The top systemic leaves that were not inoculated were collected for ELISA detection of virus content. The specific method is as follows: (1) Take 1-2g of TSWV virus source, put it in a mortar, add 5-10mL of TSWV inoculation buffer (0.1M pH7.0 phosphate buffer, sterilized at 121℃ for 20 minutes; within half an hour before inoculation, add 0.2g of sodium sulfite and 10uL of beta-mercaptoethanol to each 100mL of phosphate buffer to make TSWV inoculation buffer, and place it on ice) and 2-3g 200-400 mesh corundum is fully ground on ice until mixed evenly to obtain TSWV poison source juice; when inoculating, first sprinkle 200-400 mesh corundum evenly on the surface of the tobacco leaves to be inoculated at a dosage of 0.1-0.2g per leaf, then take TSWV poison source juice, hold the leaves of the tobacco seedlings to be inoculated with one hand, and gently and evenly rub TSWV poison source juice from the base to the tip of the leaves of the tobacco seedlings to be inoculated with the other hand; the dosage of TSWV poison source juice is 50-100ul of the inoculated leaf; (2) After rubbing, rinse the inoculated leaves with clean water, then incubate the plants in the dark at a temperature of 22-25°C and a humidity of 60-80% for one day, then move the plants to a temperature of 22-25°C, a photoperiod of 14h / 10h day / night, and a humidity of 80% for further incubation; (3) Starting from the 9th day after inoculation, fresh young tobacco leaves were taken every 7 days and tested for TSWV using a double antibody sandwich ELISA method (Agdia, Product No. SRA 39300 / 0096), and the incidence rate of tobacco seedling spotted wilt a was calculated. A total of 4 surveys were conducted. In order to reduce the error of human operation, the ELISA test of each sample was designed with three technical replicates. The operating steps of the double antibody sandwich ELISA method were carried out according to the product instructions (Agdia, Product No. SRA39300 / 0096).

[0181] The total number of diseased plants was calculated by adding the number of positive plants from the four tests. The incidence rate a = (number of susceptible plants of the variety / total number of plants of the variety) × 100%. Based on the genotyping, symptom observation after inoculation, and ELISA test results, it was found that 12 plants containing the resistance-introduced fragment (positive results for the NaChr3_59M marker test) were completely resistant to the disease, while 12 plants without the resistance-introduced fragment (negative results for the NaChr3_59M marker test) were 100% susceptible to the disease ( Figure 7 ).

[0182] Example 10. Analysis of agronomic traits of materials after removing linkage loci

[0183] Individual strain No. 12, which carries the shortest RTSW segment, was self-pollinated. Genotyping of the progeny from this self-pollinated strain was performed using the molecular marker detection primers NaChr3_59MF / NaChr3_59MR. One thousand homozygous F3 plants (genotype RTSW / RTSW) were planted in the field in 2021 (denoted as K326-RTSW in Table 9). A standard K326 variety was also planted as a control. Germination rate, survival rate, and field vigor during the seedling stage showed no difference between the K326-RTSW plants (containing the shortest RTSW segment) and the standard K326 variety. At maturity, 10 randomly selected plants were tested for agronomic traits, including plant height, stem girth, leaf number, leaf length, and leaf width, to assess the performance of the tobacco material (K326-RTSW) containing the short RTSW segment (Table 9).

[0184] Table 9 Agronomic traits analysis

[0185]

[0186] As can be seen from the table, the short RTSW introduced segment has completely eliminated linkage drag, and the obtained offspring containing the short RTSW introduced segment (K326-RTSW) have no significant differences from the main cultivated variety (K326 variety) in agronomic traits such as plant height, stem girth, number of leaves, leaf length, and leaf width. SEQUENCE LISTING <110> Yunnan Tobacco Agricultural Science Research Institute <120> Molecular markers for screening tobacco plants resistant to spotted wilt without linkage drag and their application <130> P210688-YCN <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 470 <212> DNA <213> Nicotiana alata <400> 1 ctctgcctag atgttgttaa ttgcgatcgc aggtaatcat ctgcgatcgc gaagagtaaa 60 agttaaaggc tccagaaatt gggctatgcg aacacagctt aaggaatgcc atcgcagaag 120 aggaagtagc agacatatgt gttcgcgaac agggccacgc gatcgcgaag aggaaaatgg 180 ccactgagcc accgtctcag tttctactat gcaaacgcga agaatgggat gcaaacgtga 240 aggaggaggg aatagacttc cgcgatcgcg aaccaactaa tgcgatcacg tagaacaaaa 300 tggtccccct ccagaattac tctatgcgaa tgcggaggaa gggctacgat cgcactgaag 360 gaaactagat accagctgtt cagcaattca acaagttcca aaatgatctg ttgagcattc 420 gaaacacacc cgaggccccc gggacctcaa ccaaatctac ggacatatcc 470 <210> 2 <211> 214 <212> DNA <213> Nicotiana alata <400> 2 gtggaggata cgattacgcc tgtcgcgc taggaggacg tcttatactt agagatgtat 60 gggatgccct ttcaacattt ttagaccgtt acaaccgttt caagggtgat acgccgattg 120 acggttaagg ggccattcta tggtgcctgt cataatgacg cacgttactg tatacctgaa 180 ccgttttacg gttggtcaag cagttaatga tggc 214 <210> 3 <211> 243 <212> DNA <213> Nicotiana alata <400> 3 gggtgtgttt cgggttgtga atccgctctt attcgatatt tcaatattgc ttctttggat 60 ataaaagggt actatataaa gaaaatgagc tcccaattaa gttttgttta aaccattaga 120 tccgtatcga aattcggag cataggaaa aagaatcgtc gaagttggac atcatatggg 180 gaagttatgc tcgtttccgt gccgagaaat ctggttgctg tcaaaccgtg gaagcggaaa 240 ctg 243 <210> 4 <211> 244 <212> DNA <213> Nicotiana alata <400> 4 gcacgccgtc cactttgaat gttgaatatg ggatcgtgtg gcgcgccgtc cactttgtgc 60 ttactgtttt acttagagta gtcgtagatt aactgcttta gcttagtgta ctttatgctt 120 tagaattgtt cccccttaca gcatattccc ctcccgtatc tgtttacttt acagtatttc 180 ttcctgttgt cgtatgcata tatatcttgc acaggttaac gttgtagtc ctgtctagcc 240 tcgt 244 <210> 5 <211> 250 <212> DNA <213> Nicotiana alata <400> 5 gcattgttcc gacttgtaga atccttcagg gattgatatt acagagttag catgatttgc 60 atactatttg aactcagtcc gaagttttta acactatttt gcaaactcag ccatatttct 120 aagttttgaa aactcaaatg acttttaaat gatatttcgg gctgatgatc actgttttat 180 aaatgcccaa gggcttatga cgttttctgg actgtatatt atgacttgga acagtggtaa 240 ctattggcac 250 <210> 6 <211> 200 <212> DNA <213> Nicotiana alata <400> 6 tcttacctct cctactactc ctccatctat tatttcctct tcctctgata cgccttcata 60 aacaggtccg agtaacagag gtataactat gaggaaggtt acgcttaaag ttcctgctac 120 tcatagtctt ttaaggaaga ctggtggaac tgacgtgtgg cttgagcctt taattggaga 180 tacggagaag aagaagatgc 200 <210> 7 <211> 633 <212> DNA <213> Nicotiana alata <400> 7 cttagcaggc aaccagacag gcacggtacc tggaggacat aaaatttcag ccaaccacgg 60 ccgagcttct tctttcaaag ctaacttctc caagtaccgt gtctcatcct catctccgaa 120 tcccaagaaa tcattgagag tcgtgccatc gaacggaagt cgcttgtctc ttacttgagt 180 tgtctttgta tcccgaacca aatggtgaac attggcgtaa aactccttca ccatgtgttc 240 gttggcattc tccaacttct cttaaagta cccccagccc tcacgtgtgc ggaattgggc 300 ttgcacacga gggttgtggg gaagcagatc actatctatg aacctcctct cgggtatcaa 360 ttttctgtcc ggccaccaca gcctgaattt agagtaagcc gtctcactga tgaatctatc 420 aacccatgca tttgggttct tggttctgtc gatgccccca gttgctggag caccaccatc 480 actggtactc tcttcttccc ccgatagaga tgctgtgggg aaagtggagt actctccttc 540 tccagtttca tttcatctc cggactcgtc agactggtct gagactgttc tgttctctgg 600 ccgcttggct aacacattct cttgctcatt ctc 633 <210> 8 <211> 494 <212> DNA <213> Nicotiana alata <400> 8 gctgctcaaa ctggcttatg agcattttt ggtttatcta cgcgtttggt aaataccgaa 60 agtgcttata agccagatac ttataagctt aaatcagcca taagtcataa gctgggcacc 120 atcagcctat gactttttag cttataagca ctttagttg accaagtttt ttactagttt 180 atccttaata atatttttta ttcacaaaa tattttttcc aaaataattt ttctaacttt 240 cttttcatttt catatttgtt cttcatttt ttctttaca agaaactttt taatttattg 300 tcacgaccca aaccccgctc cggtcgtgat acgccctcg tgaagacaag gccagccaaa 360 ccattacaa acacctcttt taagcagtta aacagttcat aagcagtttta atatgattt 420 aatgatatta attagcggaa gtacaaccca accagccct aatcggggtg tcacaagtca 480 cgagcatcta tag 494 <210> 9 <211> 532 <212> DNA <213> Nicotiana equipment <400> 9 agcataagg tcggaaggaa gaaagcttta tctcttagg aagattaat atcagcttca 60 accaaaatca tgtttaatgt atatggactg gaaccaatg ttaccaaag tcaatggttg 120 cagtatttta ttagtacag gggctatcat attataac agatagagag ctaattggca 180 ggttataatc cattctcatt ggcctcaaaa gtcaatagtt acatctgtta atgaataaca 240 ttgcttagta tattattatt agttaatata caagtcagtc gcttataacg cgatgatcat 300 gcaagccact cctagcaatt gcagtagttt cctattattc tgtctgtgag atgctgatga 360 tgagatactt cttgctagaa gaatatttca gtagtattca gtcgactact atattaaact 420 cgaaatgtag aggctggata ttgtatcgtt caaccaacat ttattcagaa tgggctaata 480 gaacttcttc caagataaag gaagacttag agatatgcgt catcagccgt ca 532 <210> 10 <211> 444 <212> DNA <213> Nicotiana alata <400> 10 ttgatgacct cggtgaccac ttttggctgt ggaacccctc cacaagagaa tcaatagaac 60 ttccctgtcc agaatctcaa tttaaggatt atgtatgcgg gattggatat gattctacta 120 gtgacgacta taagatcgtt gcgattaacc tagaagcgaa aatccatcag gatgtatctg 180 ttgaaattct cgcgctaaaa agtggttctt ggagaaaaaa ttgtaaatat cctattggta 240 tccaccgtgt atcgggtggt gctatagatc gtggtatgga ttctttggca tttgtacatg 300 gagcatttca ttgggttgca ttcgttggg ttggtttatc acgatgttct actatcattt 360 cacttaatat ttcaagtgag gtgttcggag agataccctt gatagagcaa atgtgcaata 420 catacaacgc caagttcatc gacc 444 <210> 11 <211> 621 <212> DNA <213> Nicotiana alata <400> 11 tgacttccat attgagaagc cgattgaaaa gtacaataga taacatttct actttgatct 60 atttaatcaa tataaattcc taaatctgca aaaaaaaaa gaaaaaaaaa gaactatatc 120 catctgaact attgttctag aatcttaacc caatattatt tattttagag atgtactagt 180 atagtacccg cgcgatgcgc ggaatatggt agaagaaaaa agtataaaaa attattatct 240 tatttttagc taaatgtaaa ttttacagat atagtttaat agacagttga cactttagtc 300 attaaaagtt tgaattcagc ttcctaatat ttatttatg tagccaaaaa tgccataagt 360 tattgtggta tgttcaatga caaaataatt aaacttaact tactataaat aatgattagt 420 catgaggata aaaaaagaat tgccgggtta tttaattcca gaatatcatt gacaatttgc 480 attagcatat atggaatccg actctaagat aacatgctat tcatttcatt tgtaacattt 540 caaaccaaca ttatggatg cagcatcctt ctcaaatgac taataaaaat ataattacag 600 gtagtcgtac agacggatct c 621 <210> 12 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> NaChr3_44.2M labeled upstream primer <400> 12 ctctgcctag atgttgttaa ttgc 24 <210> 13 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> NaChr3_44.2M labeled downstream primer <400> 13 ggatatgtcc gtagatttgg ttga 24 <210> 14 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> NaChr3_54M labeled upstream primer <400> 14 gtggaggata cgattacgcc tgtc 24 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_54M labeled downstream primer <400> 15 gccatcatta actgcttgac caacc 25 <210> 16 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> NaChr3_57M labeled upstream primer <400> 16 gggtgtgttt cgggttgtga atcc 24 <210> 17 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> NaChr3_57M labeled downstream primer <400> 17 cagtttccgc ttccacggtt tga 23 <210> 18 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> NaChr3_58M labeled upstream primer <400> 18 gcacgccgtc cactttgaat g 21 <210> 19 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> NaChr3_58M labeled downstream primer <400> 19 acgaggctag acaggaccta caa 23 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_59M labeled upstream primer <400> 20 gcattgttcc gacttgtaga atcctt 26 <210> twenty one <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_59M labeled downstream primer <400> twenty one gtgccaatag ttaccactgt tccaa 25 <210> twenty two <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_60M labeled upstream primer <400> twenty two tcttacctct cctactactc ctccatc 27 <210> twenty three <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_60M labeled downstream primer <400> twenty three gcatcttctt cttctccgta tctccaa 27 <210> twenty four <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_62.6M labeled upstream primer <400> twenty four cttagcaggc aaccagacag 20 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_62.6M labeled downstream primer <400> 25 gagaatgagc aagagaatgt gttag 25 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> NaChr3_64.6M labeled upstream primer <400> 26 gctgctcaaa ctggcttatg a 21 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> NaChr3_64.6M labeled downstream primer <400> 27 ctaatagatg ctcgtgactt gtga 24 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_65.7M labeled upstream primer <400> 28 agcataaagg tcggaaggaa gaaagc 26 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NaChr3_65.7M labeled downstream primer <400> 29 tgacggctga tgacgcatatctcta 25 <210> 30 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> NaChr4_2M labeled upstream primer <400> 30 ttgatgacct cggtgaccac tt 22 <210> 31 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> NaChr4_2M labeled downstream primer <400> 31 ggtcgatgaa cttggcgttg t 21 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NaChr4_8M labeled upstream primer <400> 32 tgacttccat attgagaagc cgattga 27 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> NaChr4_8M labeled downstream primer <400> 33 gagatccgtc tgtacgacta cctgta 26

Claims

1. A method for selecting tobacco plants or germplasm for resistance to spotted wilt with reduced linkage drag, the method comprising: (a) isolating nucleic acids from tobacco plants or germplasm; (b) detecting a spotted wilt resistance marker in the isolated nucleic acid, wherein the spotted wilt resistance marker comprises the NaChr3_59M marker shown in SEQ ID No. 5; Detecting a first-linked drag site marker and a second-linked drag site marker in the isolated nucleic acid; the first-linked drag site marker includes the NaChr4_2M marker shown in SEQ ID No. 10 and the NaChr4_8M marker shown in SEQ ID No. 11; the second-linked drag site marker includes the NaChr3_44.2M marker shown in SEQ ID No. 1, the NaChr3_54M marker shown in SEQ ID No. 2, the NaChr3_57M marker shown in SEQ ID No. 3, the NaChr3_58M marker shown in SEQ ID No. 4, the NaChr3_62.6M marker shown in SEQ ID No. 7, and the NaChr3_64.6M marker shown in SEQ ID No. 8; (c) selecting tobacco plants or germplasm comprising the spotted wilt resistance marker and not comprising the first linked drag locus marker and the second linked drag locus marker.

2. The method according to claim 1, characterized in that The tobacco plant or germplasm resistant to spotted wilt is obtained through hybrid breeding, mutation breeding, genome editing breeding and / or transgenic breeding.

3. The method according to claim 1, characterized in that The detection may include polymerase chain reaction or nucleic acid sequencing.

4. The method according to claim 1, wherein The amplification primers for the NaChr3_59M marker are shown in SEQ ID NO. 20 and SEQ ID NO. 21; The NaChr4_2M-labeled amplification primers are shown in SEQ ID No. 30 and SEQ ID No. 31; The NaChr4_8M-labeled amplification primers are shown in SEQ ID No. 32 and SEQ ID No. 33; The amplification primers for the NaChr3_62.6M label are shown in SEQ ID No. 24 and SEQ ID No. 25; The amplification primers for the NaChr3_64.6M label are shown in SEQ ID No. 26 and SEQ ID No. 27; The NaChr3_44.2M-labeled amplification primers are shown in SEQ ID No. 12 and SEQ ID No. 13; The amplification primers for the NaChr3_54M label are shown in SEQ ID No. 14 and SEQ ID No. 15; The amplification primers for the NaChr3_57M label are shown in SEQ ID No. 16 and SEQ ID No. 17; The NaChr3_58M-labeled amplification primers are shown in SEQ ID No. 18 and SEQ ID No.

19.

5. A method for developing tobacco plants or germplasm with reduced linkage drag that is resistant to spotted wilt, the method comprising: (a) hybridizing a first tobacco plant or its germplasm with a second tobacco plant or its germplasm to produce a progeny tobacco plant or germplasm thereof, wherein the first tobacco plant or its germplasm comprises a spotted wilt resistance marker and does not comprise a first linked drag site marker and a second linked drag site marker; the spotted wilt resistance marker comprises the NaChr3_59M marker set forth in SEQ ID No. 5; the first linked drag site marker comprises the NaChr4_2M marker set forth in SEQ ID No. 10 and the NaChr4_8M marker set forth in SEQ ID No. 11; the second linked drag site marker comprises the NaChr3_44.2M marker set forth in SEQ ID No. 1, the NaChr3_54M marker set forth in SEQ ID No. 2, the NaChr3_57M marker set forth in SEQ ID No. 3, the NaChr3_58M marker set forth in SEQ ID No. 4, the NaChr3_62.6M marker set forth in SEQ ID No. 7, and the NaChr3_64.6M marker set forth in SEQ ID No. 8; (b) isolating nucleic acid from the progeny tobacco plant or germplasm; (c) detecting the spotted wilt resistance marker, the first linked drag locus marker, and the second linked drag locus marker in the isolated nucleic acid, thereby producing a progeny tobacco plant or germplasm that comprises the spotted wilt resistance marker and does not comprise the first linked drag locus marker and the second linked drag locus marker. 6 . The method according to claim 5 , wherein the first tobacco plant or germplasm thereof is obtained by a hybrid breeding method, a mutagenesis breeding method, a genome editing breeding method and / or a transgenic breeding method. The method of claim 5 , wherein the detecting comprises polymerase chain reaction or nucleic acid sequencing.

8. The method according to any one of claims 5 to 7, wherein the first tobacco plant or its germplasm and the second tobacco plant or its germplasm are selected from Burley type, dark type, flue-cured type, Maryland type, Oriental type or cigar type.

9. A molecular marker set for screening tobacco plants or germplasm with reduced linkage drag for resistance to spotted wilt, characterized in that: It includes a spotted wilt resistance marker, a first-linked drag site marker and a second-linked drag site marker; the spotted wilt resistance marker includes the NaChr3_59M marker shown in SEQ ID No. 5; the first-linked drag site marker includes the NaChr4_2M marker shown in SEQ ID No. 10 and the NaChr4_8M marker shown in SEQ ID No. 11; the second-linked drag site marker includes the NaChr3_44.2M marker shown in SEQ ID No. 1, the NaChr3_54M marker shown in SEQ ID No. 2, the NaChr3_57M marker shown in SEQ ID No. 3, the NaChr3_58M marker shown in SEQ ID No. 4, the NaChr3_62.6M marker shown in SEQ ID No. 7 and the NaChr3_64.6M marker shown in SEQ ID No.

8.

10. A primer set for screening tobacco plants or germplasm with reduced linkage drag and resistance to spotted wilt, characterized in that: include: Primers for amplifying the NaChr3_59M marker as shown in SEQ ID NO. 20 and SEQ ID NO. 21; Primers for amplifying the NaChr4_2M marker as shown in SEQ ID No. 30 and SEQ ID No. 31; Primers for amplifying the NaChr4_8M marker as shown in SEQ ID No. 32 and SEQ ID No. 33; Primers for amplifying the NaChr3_44.2M marker as shown in SEQ ID No. 12 and SEQ ID No. 13; Primers for amplifying the NaChr3_54M marker as shown in SEQ ID No. 14 and SEQ ID No. 15; Primers for amplifying the NaChr3_57M marker as shown in SEQ ID No. 16 and SEQ ID No. 17; Primers for amplifying the NaChr3_58M marker as shown in SEQ ID No. 18 and SEQ ID No. 19; Primers for amplifying the NaChr3_62.6M marker as shown in SEQ ID No. 24 and SEQ ID No. 25; and SEQ ID No. 26 and SEQ ID No. 27 are primers for amplifying the NaChr3_64.6M marker.

11. A method for screening tobacco plants or germplasm for resistance to spotted wilt, the method comprising: (a) isolating nucleic acids from tobacco plants or germplasm; (b) detecting at least one spotted wilt resistance marker in the isolated nucleic acid; The spotted wilt resistance marker includes the NaChr3_59M marker shown in SEQ ID No. 5; (c) selecting tobacco plants or germplasm comprising at least one spotted wilt resistance marker.

12. The method according to claim 11, characterized in that The detection may include polymerase chain reaction or nucleic acid sequencing.

13. The method according to claim 12, characterized in that The NaChr3_59M-labeled amplification primers are shown in SEQ ID NO. 20 and SEQ ID NO.

21.

14. A molecular marker for screening tobacco plants or germplasm resistant to spotted wilt, the nucleotide sequence of which is shown in SEQ ID NO.

5.

15. The molecular marker according to claim 14, characterized in that The nucleotide sequences of the primers used to amplify the molecular markers are shown in SEQ ID NO. 20 and SEQ ID NO. 21.

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