Tobacco Plant Resistant To Spotted Wilt Disease Without Linkage Drag And Method For Breeding The Same

Abstract

The invention relates to the field of tobacco breeding, particularly to a tobacco plant resistant to TSWD without linkage drag and a method for breeding the same. Provided is a tobacco plant or germplasm resistant to TSWD, which comprises a short RTSW introgressed segment, wherein at least part or entire of the sequence set forth in SEQ ID No. 34 is deleted in the short RTSW introgressed segment as compared to the RTSW introgressed segment of tobacco Polata. Also provided is a method for screening said tobacco plant or germplasm, in which a tobacco plant or germplasm resistant to TSWD carrying a short RTSW introgressed segment is obtained by detecting NaChr4_2M, NaChr4_8M, NaChr3_62.6M and NaChr3_64.6M linkage drag locus markers. Compared with Polalta, the tobacco plant or germplasm provided by the invention not only has TSWD resistance, but also reduces or removes linkage drag.

Claims

1. A tobacco plant or germplasm resistant to TSWD, which comprises a short RTSW introgressed segment, wherein at least part or entire of the sequence set forth in SEQ ID No. 34 is deleted in the short RTSW introgressed segment as compared to the RTSW introgressed segment of tobacco Polata.

2. The tobacco plant or germplasm according to claim 1, wherein the tobacco plant or germplasm does not comprise a first linkage drag locus marker and/or a second linkage drag locus marker; the first linkage drag locus marker comprises NaChr4_2M marker set forth in SEQ ID No. 10 and/or NaChr4_8M marker set forth in SEQ ID No. 11; the second linkage drag locus marker comprises NaChr3_62.6 M marker set forth in SEQ ID No. 7 and/or NaChr3_64.6 M marker set forth in SEQ ID No. 8; the NaChr3_62.6 M marker is located at 1-633 bp of the sequence set forth in SEQ ID No. 34; the NaChr3_64.6 M marker is located at 2210450-2210943 bp of the sequence set forth in SEQ ID No. 34.

3. The tobacco plant or germplasm according to claim 2, wherein the second linkage drag locus marker further comprises NaChr344.2M marker set forth in SEQ ID No. 1, NaChr3_54M marker set forth in SEQ ID No. 2, NaChr3_57M marker set forth in SEQ ID No. 3 and/or NaChr3_58M marker set forth in SEQ ID No. 4.

4. The tobacco plant or germplasm according to claim 2, wherein the tobacco plant or germplasm comprises a TSWD resistance marker that comprises NaChr3_59M marker set forth in SEQ ID No. 5.

5. The tobacco plant or germplasm according to claim 4, wherein the tobacco plant or germplasm comprises NaChr3_59 M marker set forth in SEQ ID No. 5, and does not comprise NaChr4_2 M marker set forth in SEQ ID No. 10, NaChr4_8M marker set forth in SEQ ID No. 11, and NaChr3_64.6M marker set forth in SEQ ID No. 8.

6. The tobacco plant or germplasm according to claim 1, wherein the short RTSW introgressed segment is obtained by means of chromosome recombination, genome editing, chemical mutagenesis, physical mutagenesis, or artificial de novo gene synthesis.

7. The tobacco plant or germplasm according to claim 1, wherein the tobacco plant or germplasm is selected from Burley tobacco, Dark tobacco, Flue-cured tobacco, Maryland tobacco, Oriental tobacco or Cigar tobacco.

8. A method for screening a tobacco plant or germplasm resistant to TSWD with reduced linkage drag, the method comprising: (a) screening tobacco plants or germplasms resistant to TSWD and isolating their nucleic acids; (b) detecting a first linkage drag locus marker and/or a second linkage drag locus marker in the isolated nucleic acids; the first linkage drag locus marker comprises NaChr4_2 M marker set forth in SEQ ID No. 10 and/or NaChr4_8M marker set forth in SEQ ID No. 11; the second linkage drag locus marker comprises NaChr3_62.6M marker set forth in SEQ ID No. 7 and/or NaChr3_64.6M marker set forth in SEQ ID No. 8; (c) selecting a tobacco plant or germplasm resistant to TSWD that does not comprise the first linkage drag locus marker and/or the second linkage drag locus marker.

9. The method according to claim 8, wherein the method for screening a tobacco plant or germplasm resistant to TSWD comprises: isolating nucleic acids of a tobacco plant or germplasm; detecting a TSWD resistance marker in the isolated nucleic acids; the TSWD resistance marker comprises NaChr3_59 M marker set forth in SEQ ID No. 5; selecting a tobacco plant or germplasm comprising the TSWD resistance marker.

10. The method according to claim 8, wherein the tobacco plant or germplasm resistant to TSWD is obtained by hybrid breeding methods, mutation breeding methods, genome editing breeding methods, and/or transgenic breeding methods.

11. The method according to claim 8, wherein the second linkage drag locus marker further comprises NaChr344.2M marker set forth in SEQ ID No. 1, NaChr3_54M marker set forth in SEQ ID No. 2, NaChr3_57M marker set forth in SEQ ID No. 3, and/or NaChr3_58M marker set forth in SEQ ID No. 4.

12. The method according to claim 8, wherein the detection includes polymerase chain reaction or nucleic acid sequencing.

13. A method for breeding a tobacco plant or germplasm resistant to TSWD with reduced linkage drag, the method comprising: (a) crossing a first tobacco plant or germplasm thereof with a second tobacco plant or germplasm thereof to produce a progeny tobacco plant or germplasm thereof, wherein the first tobacco plant or germplasm thereof comprises a TSWD resistance marker and does not comprise a first linkage drag locus marker and/or a second linkage drag locus marker; the TSWD resistance marker comprises NaChr3_59M marker set forth in SEQ ID No. 5; the first linkage drag locus marker comprises NaChr4_2M marker set forth in SEQ ID No. 10 and/or NaChr4_8M marker set forth in SEQ ID No. 11; the second linkage drag locus marker comprises NaChr3_62.6M marker set forth in SEQ ID No. 7 and/or NaChr3_64.6M marker set forth in SEQ ID No. 8; (b) isolating nucleic acids from the progeny tobacco plant or germplasm thereof; (c) detecting the TSWD resistance marker, the first linkage drag locus marker, and the second linkage drag locus marker in the isolated nucleic acids, thereby producing a progeny tobacco plant or germplasm that comprises the TSWD resistance marker and does not comprise the first linkage drag locus marker and/or the second linkage drag locus marker.

14. The method according to claim 13, wherein the first tobacco plant or germplasm thereof is obtained by hybrid breeding methods, mutation breeding methods, genome editing breeding methods and/or transgenic breeding methods.

15. The method according to claim 13, wherein the second linkage drag locus marker further comprises NaChr344.2M marker set forth in SEQ ID No. 1, NaChr3_54M marker set forth in SEQ ID No. 2, NaChr3_57M marker set forth in SEQ ID No. 3, and/or NaChr3_58M marker set forth in SEQ ID No. 4.

16. The method according to claim 13, wherein the detection includes polymerase chain reaction or nucleic acid sequencing.

17. The method according to claim 13, wherein the first tobacco plant or germplasm thereof and the second tobacco plant or germplasm thereof are selected from Burley tobacco, Dark tobacco, Flue-cured tobacco, Maryland tobacco, Oriental tobacco or Cigar tobacco.

18-20. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0085] FIG. 1 shows the phenotype of linkage drag of tobacco plants carrying TSWD resistance locus. Tobacco plants were obtained by crossing the tobacco variety Polalta as the donor parent and the susceptible elite cultivar 1(326 (N tabacum cv. K326) as the recipient parent, and backcrossing for 5 generations using 1(326 as the recurrent parent. FIG. 1A shows the phenotype of leaf deformations, including thickened veins, irregular distortions of veins, thickened leaves and veins, and narrow leaves; FIGS. 1B and 1C show the deformed phenotypes of the whole plant, which are characterized by growth retardation, distortions of stems, and dwarfing of plants.

[0086] FIG. 2 shows the distribution of SNP sites between resistant pool (RTSW-pool) and susceptible pool (rtsw-pool) on chromosome 3 of N alata genome from 43.7 Mb to 64.8 Mb (Chr3: 43.7-64.8 Mb) that is found by analysis using algorithms of delta SNP-index and Euclidean distance (ED).

[0087] FIG. 3 shows the analysis results of deformed susceptible pool (DEF_rtsw_pool) and normal susceptible pool (def_rtsw_pool) using algorithms of delta SNP-index and Euclidean distance (ED). By Bulked Segregant Analysis (BSA), it was found that the DEF1 locus causing deformations was located at the end of chromosome 4 in the range of 1-8.6 Mb.

[0088] FIG. 4 shows the predicted genetic models of DEF1 and RTSW (DEF2) loci. DEF1 locus is derived from 1-8.6 Mb of chromosome 4 of wild tobacco N alata. DEF2 locus is tightly linked to the resistance locus (RTSW locus). They are located at the same locus that is derived from 43.7 Mb to 64.8 Mb of chromosome 3 of N alata. It is speculated that there may be a tightly linked toxin gene (Toxin) near the RTSW (DEF2) locus, and the existence of RTSW (DEF2) locus alone may lead to heavier deformed phenotype or impaired fertility, while DEF1 locus may be tightly linked to a detoxin gene (Detoxin), and only in the presence of DEF1 locus, RTSW (DEF2) locus can be inherited stably. The markers used in the invention have been marked in the figure.

[0089] FIG. 5 shows part of the results of screening using molecular markers NaChr3_59M and NaChr3_64.6 M located at both ends of the resistance introgressed segment of plant No. 46. Molecular markers at both ends of the RTSW (DEF2) locus were used simultaneously to screen. Using an optimized molecular marker screening system, 18 plants comprising at least one of the molecular markers NaChr3_59M and NaChr3_64.6 M were obtained.

[0090] FIG. 6 shows the phenotype in the rosette stage and the adult stage. A, 5 plants, carrying RTSW resistance locus and breaking linkage drag, obtained from 160,000 tobacco screening. B, a typical TSWD resistance tobacco with linkage drag. C, the adult plant phenotype of plant No. 12; its main agronomic traits such as plant height, leaves and flowering period are the same as those of the elite cultivar K326.

[0091] FIG. 7 shows virus inoculation test of self-crossed progenies of plant No. 12. Self-crossed progenies of plant No. 12 were genotyped using amplification primers NaChr3_59MF/NaChr3_59MR for NaChr3_59M molecular marker. Plants that comprise the molecular marker (marked as RTSW in the figure) and those that do not comprise the molecular marker (marked as rtsw in the figure) were selected and inoculated with Tomato spotted wilt virus (TSWV) by rubbing, and TSWV symptoms were observed at two weeks postinoculation. It was found that the plants carrying the resistance introgressed segment were completely resistant to the disease, and the plants without the resistance introgressed segment were 100% diseased.

DESCRIPTION OF THE SEQUENCE LISTING

[0092] SEQ ID No. 1 is the nucleotide sequence of NaChr344.2M marker. [0093] SEQ ID No. 2 is the nucleotide sequence of NaChr3_54 M marker. [0094] SEQ ID No. 3 is the nucleotide sequence of NaChr3_57 M marker. [0095] SEQ ID No. 4 is the nucleotide sequence of NaChr3_58 M marker. [0096] SEQ ID No. 5 is the nucleotide sequence of NaChr3_59 M marker. [0097] SEQ ID No. 6 is the nucleotide sequence of NaChr3_60 M marker. [0098] SEQ ID No. 7 is the nucleotide sequence of NaChr3_62.6 M marker. [0099] SEQ ID No. 8 is the nucleotide sequence of NaChr3_64.6 M marker. [0100] SEQ ID No. 9 is the nucleotide sequence of NaChr3_65.7 M marker. [0101] SEQ ID No. 10 is the nucleotide sequence of NaChr4_2M marker. [0102] SEQ ID No. 11 is the nucleotide sequence of NaChr4_8M marker. [0103] SEQ ID No. 12 is the forward primer used to amplify the NaChr344.2M marker. [0104] SEQ ID No. 13 is the reverse primer used to amplify the NaChr344.2M marker. [0105] SEQ ID No. 14 is the forward primer used to amplify the NaChr3_54 M marker. [0106] SEQ ID No. 15 is the reverse primer used to amplify the NaChr3_54 M marker. [0107] SEQ ID No. 16 is the forward primer used to amplify the NaChr3_57 M marker. [0108] SEQ ID No. 17 is the reverse primer used to amplify the NaChr3_57 M marker. [0109] SEQ ID No. 18 is the forward primer used to amplify the NaChr3_58 M marker. [0110] SEQ ID No. 19 is the reverse primer used to amplify the NaChr3_58 M marker. [0111] SEQ ID No. 20 is the forward primer used to amplify the NaChr3_59 M marker. [0112] SEQ ID No. 21 is the reverse primer used to amplify the NaChr3_59 M marker. [0113] SEQ ID No. 22 is the forward primer used to amplify the NaChr3_60 M marker. [0114] SEQ ID No. 23 is the reverse primer used to amplify the NaChr3_60 M marker. [0115] SEQ ID No. 24 is the forward primer used to amplify the NaChr3_62.6 M marker. [0116] SEQ ID No. 25 is the reverse primer used to amplify the NaChr3_62.6 M marker. [0117] SEQ ID No. 26 is the forward primer used to amplify the NaChr3_64.6 M marker. [0118] SEQ ID No. 27 is the reverse primer used to amplify the NaChr3_64.6 M marker. [0119] SEQ ID No. 28 is the forward primer used to amplify the NaChr3_65.7 M marker. [0120] SEQ ID No. 29 is the reverse primer used to amplify the NaChr3_65.7 M marker. [0121] SEQ ID No. 30 is the forward primer used to amplify the NaChr4_2M marker. [0122] SEQ ID No. 31 is the reverse primer used to amplify the NaChr4_2M marker. [0123] SEQ ID No. 32 is the forward primer used to amplify the NaChr4_8 M marker. [0124] SEQ ID No. 33 is the reverse primer used to amplify the NaChr4_8 M marker. [0125] SEQ ID No. 34 is a DNA sequence on the RTSW introgressed segment of Polalta containing the second linkage drag locus (DEF2) but not the RTSW gene.

DETAILED DESCRIPTION OF THE INVENTION

[0126] The invention will be further described in detail below by examples. Those skilled in the art will understand that the following examples are only used to illustrate the invention and should not be regarded as limiting the scope of the invention. For those examples in which specific technologies or conditions are not indicated, it shall be carried out in accordance with the technologies or conditions described in the literature in the art or in accordance with the product specification. The reagents or instruments used without an indication of the manufacturer are all conventional products that can be obtained through purchase.

[0127] Tobacco material Polalta is a TSWV resistant tobacco material carrying the RTSW locus (TSWD resistance locus), which has been published in a non-patent literature (Laskowska D, Berbe A, 2010. TSWV resistance in DH lines of tobacco (N tabacum L.) obtained from a hybrid between Polalta and Wilica. Plant Breeding 129, 731-3.). Tobacco variety 1(326 (N tabacum cv. K326) is a TSWV-susceptible tobacco material that does not possess the RTSW locus (TSWD resistance locus), which has been published in a non-patent literature (Edwards et al., 2017, A reference genome for Nicotiana tabacum enables map-based cloning of homologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448.), and the public can obtain its reference genome sequence from the website of https://solgenomics.net/organism/Nicotiana tabacum/genome. BC5F3, BC6F1 and BC6F2 progeny populations obtained by crossing of Polalta and 1(326, backcrossing and self-crossing were created and preserved by our research group. N alata is a kind of wild tobacco resistant to TSWV, which has been published in a non-patent literature (Laskowska et al., 2013, A survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus (TSWV). Euphytica 193, 207-19.). N alata used in the invention has an accession number of PI42334 in the tobacco germplasm bank of the United States. The above tobacco materials are common tobacco germplasm resources, and the public can obtain them from tobacco germplasm resources preservation organizations or Yunnan Academy of Tobacco Agricultural Sciences.

[0128] TSWV and Agrobacterium EHA105 carrying the avirulence gene NSm for resistance test are preserved in Yunnan Academy of Tobacco Agricultural Sciences. For the preparation method of Agrobacterium EHA105 carrying the avirulence gene NSm, see Chinese patent no. ZL201710414755.X, titled A Method for Identifying Tobacco Resistance Using the Tomato Spotted Wilt Virus NSm Gene. The entire content of this patent is incorporated herein by reference.

[0129] Reagents: All molecular biology-related reagents, instruments and consumables are commercially available products.

Example 1. Acquisition of BC6F2 Segregation Population and Definition of TSWD Resistance with Linkage Drag

[0130] The material Polalta (5) carrying RTSW spotted wilt disease resistance locus was used as the donor parent to cross with the main susceptible variety 1(326 (?) (N tabacum cv. K326) as the recipient parent, and the progeny backcrossed with the recurrent parent 1(326 for 5 generations. Homozygous tobacco backcrossing and self-crossing generation BC5F3 was obtained by selecting disease resistance traits. After multiple generations of backcrossing, the resistance to TSWD was stable, but the deformed phenotypes still existed without any elimination. Deformed phenotypes included growth retardation, dwarfing of plants, and leaf deformations including thickened veins, irregular distortions of veins, thickened leaves and veins, narrow leaves, etc., as well as varying degrees of reduced fertility, such as decreased seed setting rate, shriveled fruit and decreased seed quantity of a single fruit (FIG. 1). The homozygous BC5F3 tobacco was used as the donor parent and the main susceptible variety 1(326 was used as the recurrent parent to continue backcrossing to obtain BC6F1 population, and BC6F1 population was self-crossed to obtain BC6F2 population. Using the avirulence gene induced HR method established by our team, 454 plants in the BC6F2 population were subjected to TSWD resistance identification. The avirulence gene induced HR method was the method for identifying tobacco resistance using the TSWV NSm gene that was recorded in the Chinese patent no. ZL201710414755.X, titled A Method for Identifying Tobacco Resistance Using the Tomato Spotted Wilt Virus NSm Gene. The specific steps were as follows: [0131] (1) culturing Agrobacterium EHA105 carrying the avirulence gene NSm expression vector in Agrobacterium tumefaciens medium LB at 28 C. for 24 hours, collecting bacterial cells by centrifugation, and then diluting the bacterial cells with infiltration buffer (10 mmol/L MgCl.sub.2, 10 mmol/L MES, 200 mol/L acetosyringone) to obtain a bacterial suspension with an OD.sub.600 of 0.5; [0132] (2) using a sterile syringe without needle to inject 9.5-10.5 microliters of the bacterial suspension between leaf veins from the leaf back of a tobacco plant to form a visible infiltration zone; inoculated tobacco plants were cultured with 20-28 C., 80% humidity and 16/8 light cycles for 72 hours. [0133] (3) observing; if the tobacco plant showed typical hypersensitive reaction (HR) induced by the identified strain carrying the avirulence gene NSm expression vector, the tobacco plant was identified as a disease-resistant plant relative to the avirulent gene NSm.

[0134] By NSm mediated disease resistance identification, among the 454 plants of BC6F2 population, 319 plants showed HR phenotype and were resistant plants (indicated by RTSW), while 118 plants did not show HR phenotype and were susceptible plants (indicated by rtsw), and the other 17 plants were too small for HR test, so they were not included in the statistics.

[0135] Next, the genetic relationship of TSWD resistance locus was further verified using the HR test results. A chi-square test was performed on the resistance traits of the BC6F2 population. Under the premise that the expected ratio was 3:1, .sup.2=0.9344, when df=1, .sup.20.05=0.3337, the test result .sup.2>0.3337, indicating it was not a significant difference. It was proved that the segregation of resistance traits in the BC6F2 population conformed to Mendel's law of inheritance. By chi-square test, it can be determined that RTSW gene-mediated resistance to TSWD is a single dominant gene (see Table 1 for the results).

TABLE-US-00001 TABLE 1 Segregation ratio of resistance traits in BC6F2 population HR No HR Generation (Resistant, (Susceptible, Expected Chi-square Degree of Progressive (Population) RTSW) rtsw) ratio value freedom (df) significance BC6F2 319 118 3:1 0.9344 1 0.333722

[0136] Meanwhile, phenotype observation and statistical analysis of deformity was carried out. Based on the deformed phenotypes, the genetic relationship of the deformity locus was further verified. In the statistical process of deformed phenotypes, all abnormal leaves and plant development phenotypes were classified as deformed phenotypes and represented by DEF (Deformity), and phenotypes that plant development was completely normal without any deformations were classified as normal phenotypes and represented by def. A chi-square test was performed on the resistance traits of the BC6F2 population. The deformity (DEF): the normal (def) was 417:37=11.27:1, which was much higher than 3:1 and closer to a segregation ratio of 15:1. Under the premise that the expected ratio was 15:1, .sup.2=2.79, when df=1, .sup.2.sub.0.05=0.094, the test result .sup.2>0.094, indicating that the difference was not significant. It was proved that the deformity traits in the BC6F2 population conformed to Mendel's law of inheritance, and was possibly determined by two loci.

[0137] The subpopulation BC6F2:S consisting of all susceptible plants in the BC6F2 population was also analyzed. There were 118 susceptible plants in the BC6F2:S population, and among them the deformity (DEF): the normal (def) was 81:37=2.18:1, which was closer to a segregation ratio of 3:1. Therefore, under the premise that the expected ratio was 3:1, .sup.2=2.54, when df=1, .sup.2.sub.005=0.111, the test result .sup.2>0.111, indicating that the difference was not significant, and it was proved that the susceptible sub-population BC6F2: S conformed to Mendel's single-gene inheritance law (see Table 2 for the results).

TABLE-US-00002 TABLE 2 Segregation ratio of deformed phenotypes in BC6F2 population Generation Expected Chi-square Degree of Progressive (population) Deformity Normal ratio value freedom (df) significance BC6F2 417 37 15:1 2.7964 1 0.094472 BC6F2: S 81 37 3:1 2.542 1 0.111 (susceptible)

[0138] The results of phenotype analysis and chi-square test showed that the deformed phenotypes in the population were controlled by multiple genes, probably by two loci.

Example 2. Preliminary Localization of TSWD Resistance Locus and Molecular Marker Development

[0139] In order to eliminate the interference of deformed phenotypes on localization of TSWD resistance locus, 40 deformed plants were randomly selected from 319 resistant plants to construct a resistant pool (RTSW-pool) and 40 deformed plants were randomly selected from 118 susceptible plants to construct a susceptible pool (rtsw-pool). 0.1 g leaves from each of the selected 40 plants were taken, and a total of 4 g leaves were mixed to form the RTSW-pool or rtsw-pool. The DNA of each pool was sequenced. The resistant parent Polalta and the susceptible parent K326 were also sequenced. The sequencing depth was 30, and 135G data was sequenced for each sample. Sequencing was performed using the BGI500 sequencing platform of BGI. The clean reads of the resistant pool and the susceptible pool obtained by whole genome sequencing were mapped to the chromosome level genome of Nalata respectively and analyzed by Bulked Segregant Analysis (BSA). Using delta SNP-index, a total of 7774 SNP sites between the resistant pool and the susceptible pool with a Delta value greater than 0.5 and a significance greater than 99% were found, of which 7732 SNP sites were distributed on chromosome 3 of the Nalata genome from 43.7 Mb to 64.8 Mb (Chr3: 43.7-64.8 Mb). The same result was also obtained by using Euclidean distance (ED) algorithm (see FIG. 2).

Example 3. Development and Verification of Molecular Markers of TSWD Resistance Locus

[0140] According to the SNP distribution obtained from the BSA data of the resistant pool/susceptible pool, we believe that the resistance introgressed segment in the TSWD resistant tobacco in the BC6 population is derived from chromosome 3 of the Nalata genome from 43.7 Mb to 64.8 Mb, and its size is 21.1 Mb. In order to further decrease the size of the introgressed segment, molecular markers near the left end and the right end of the introgressed segment were developed (Table 3).

TABLE-US-00003 TABLE3 Molecularmarkersoftheresistance introgressedsegment(1) Positionofthe Primer markerinthe Marker Primer sequence introgressed name name (5'-3') segment NaChr3_ NaChr3_ CTCTGCCTAGAT Atthe 44.2M 44.2MF GTTGTTAATTGC leftend (SEQIDNo.12) NaChr3_ GGATATGTCCGT 44.2MR AGATTTGGTTGA (SEQIDNo.13) NaChr3_ NaChr3_ GCTGCTCAAACT Atthe 64.6M 64.6MF GGCTTATGA rightend (SEQIDNo.26) NaChr3_ CTAATAGATGCT 64.6MR CGTGACTTGTGA (SEQIDNo.27) NaChr3_ NaChr3_ AGCATAAAGGTC Onthe 65.7M 65.7MF GGAAGGAAGAAAGC flankof (SEQIDNo.28) theright end NaChr3_ TGACGGCTGATG 65.7MR ACGCATATCTCTA (SEQIDNo.29)

[0141] In order to verify the validity of the markers, N alata, TSWD resistant plant Polalta, TSWD susceptible variety 1(326, BC6F1 population and 437 plants of BC6F2 that have been identified as resistant or susceptible to TSWD were selected for marker verification. The method of marker verification was as follows: Leaf genomic DNA was extracted from a tobacco plant to be tested using Plant Genomic DNA Extraction Kit (TIANGEN Biotech, Cat. no. DP360) according to the operating steps described in the manual of the kit. For each marker, PCR amplification was performed using the leaf genomic DNA of the tobacco plant to be tested as a template and a primer pair consisting of two single-stranded DNA. The PCR reaction system was as follows: 2Premix Ex TaqMix PCR Buffer (Takara, Cat. no. RR003A) 12.5 L, 10 mol/L forward primer 0.5 L, 10 mol/L reverse primer 0.5 L, 50 ng/L template DNA 1 L, adding sterile double distilled water to a total volume of 25 L. The PCR reaction procedure was as follows: pre-denaturation for 5 min at 94 C.; followed by 35 cycles: denaturation for 30 s at 94 C., annealing for 30 s at 55 C., extension for 30 s at 72 C. and a final elongation step for 10 min at 72 C. After reaction, the PCR product was identified by electrophoresis using ZAG DNA analyzer system (Agilent, M5320AA). If the expected PCR product was detected, the test result of the marker was positive; if the expected PCR product was not detected, the test result of the marker was negative. The above-mentioned method of marker verification is applicable to verification or detection of all the markers disclosed herein.

[0142] The results showed that NaChr3_65.7M marker was positive in N alata and Polalta, but negative in other tobacco plants 1(326, BC6F1 and BC6F2 populations. NaChr344.2M and NaChr3_64.6M markers were positive in tobacco Polalta, N alata, BC6F1 population and 319 BC6F2 tobacco plants resistant to TSWD, but negative in 1(326 and 118 susceptible BC6F2 tobacco plants. These markers are tightly linked to TSWD resistance, and co-segregate with TSWD resistance phenotype in each generation. Moreover, these markers are located at both ends of the resistance introgressed segment and can be used for the later detection of shortening of the resistance introgressed segment.

Example 4. Preliminary Localization of Linkage Drag Loci and Molecular Marker Development

[0143] By resistance screening and deformed phenotype observation, the BC6F2 population can be preliminarily divided into three subpopulations: deformed resistant (genotype: DEF_RTSW), deformed susceptible (genotype: DEF_rtsw) and normal susceptible (genotype: def_rtsw). No plants with both normal phenotype and disease resistance (genotype: def RTSW) were found. 40 plants were selected from each of the three subpopulations to construct a deformed resistant pool (DEF_RTSW_pool), a deformed susceptible pool (DEF_rtsw_pool) and a normal susceptible pool (def_rtsw_pool) respectively. The construction method was taking 0.1 g leaves from each of the selected 40 plants, and mixing a total of 4 g to form a pool for DNA resequencing. The sequencing depth was 30, and 135G data was sequenced for each pool.

[0144] After the previous genetic analysis of the segregating population, it was preliminarily determined that the deformed phenotypes were determined by two dominant loci. For accurate localization of the first deformity locus (DEF1), the deformed susceptible pool (DEF_rtswpool) and the normal susceptible pool (def_rtswpool) were first selected for analysis to exclude the interference of the TSWD resistance locus. The whole genome sequencing data of the two pools and the parents was aligned to the genome of N alata, and delta SNP-Index and ED analysis was performed (FIG. 3). Through SNP typing, it was found that the deformity locus DEF1 was located in the range of 1-8.6 Mb at the end of chromosome 4 of the N alata genome. According to the whole genome sequencing results, two markers respectively near the left end and right end of the introgressed segment were developed (Table 4), which were located at 2 Mb and 8 Mb on chromosome 4 of the N alata genome.

TABLE-US-00004 TABLE4 Molecularmarkersatbothendsforthe firstdeformitylocus(DEF1)introgressed segment Positionofthe Primer markerinthe Primer sequence introgressed Markername name (5'-3') segment NaChr4_2M NaChr4_ TTGATGAC Atthe 2MF CTCGGTGA leftend CCACTT (SEQID No.30) NaChr4_ GGTCGATG 2MR AACTTGGC GTTGT (SEQID No.31) NaChr4_8M NaChr4_ TGACTTCCA Atthe 8MF TATTGAGAA rightend GCCGATTGA (SEQID No.32) NaChr4_ GAGATCCGT 8MR CTGTACGAC TACCTGTA (SEQID No.33)

[0145] Using these markers and combined with TSWD resistance identification, Polalta, N alata, 1(326, BC6F1, and 118 BC6F2 tobacco plants that were deformed susceptible (DEF_rtsw) and normal susceptible (def_rtsw) were selected for marker verification. The method of marker verification was the same as shown in Example 3.

[0146] The results showed that in N alata, Polalta, BC6F1 and 81 deformed susceptible (DEF_rtsw) BC6F2 tobacco plants, the test results of NaChr4_2M and NaChr4_8 M markers were positive, which were completely consistent with the deformed phenotype; and in 1(326 and 37 BC6F2 tobacco plants that were normal susceptible (def_rtsw), the test results of the markers were negative, which were consistent with the normal phenotype. The NaChr4_2M and NaChr4_8 M markers are located at both ends of the resistance introgressed segment and are completely linked to the DEF1 locus, so they can be used for the detection of the DEF1 locus.

Example 5. Determination of the Second Deformity Locus (DEF2)

[0147] In order to find the second deformity locus, the obtained molecular markers (NaChr4_2M and NaChr4_8 M) located at both ends of the first deformity locus (DEF1) introgressed segment were used to screen plants with deformed phenotypes, and tobacco plants that did not possess the DEF1 introgressed segment were selected. 40 plants were randomly selected from the tobacco plants that did not possess the DEF1 introgressed segment to form a mixed pool (DEF2 pool) for whole genome sequencing. BSA was performed on the obtained whole genome sequencing data and the completely normal mixed pool (def pool). Delta SNP-Index and ED analysis was performed. The clean reads of DEF2 pool and def pool obtained by whole genome sequencing were mapped to the N. alata genome, and it was found that the deformity locus DEF2 and the resistance locus (RTSW) were in the same region. Both DEF2 and RTSW loci were located on chromosome 3 of the Nalata genome from 43.7 Mb to 64.8 Mb, suggesting that the DEF2 locus and the RTSW locus overlapped. 319 deformed plants of the BC6F2 population that did not possess the first deformity locus (DEF1) were detected using the molecular markers at both ends of the resistance introgressed segment, and the marker detection results were completely consistent with the deformed phenotype, indicating that the DEF2 locus should be tightly linked to the RTSW locus and located near the RTSW locus.

Example 6. Screening of Plants with Short Resistance Introgressed Segment

[0148] Primers NaChr344.2MF/NaChr344.2MR of the marker at the left end of the resistance introgressed segment and primers NaChr3_64.6MF/NaChr3_64.6MR of the marker at the right end of the resistance introgressed segment were used for screening from a backcrossing population. The heterozygous (RTSW/rtsw) plant of BC5F2 (BC5F2-4Q) as donor parent was crossed with the recurrent parent 1(326 to produce BC6F1 backcross population. 1500 plants of the BC6F1 population were recruited for marker screening. If both markers are negative or positive, these plants were non-recombination individuals. If one marker is positive and another marker is negative, these plants were recombination events. For the primer pair NaChr344.2MF/NaChr344.2MR, the resistant control N alata and Polalta generated a 470 bp amplified product, and the susceptible control 1(326 had no amplification, indicating that the PCR amplification was specific. The primer pair NaChr3_64.6MF/NaChr3_64.6MR was also used for amplification, the resistant control N alata and Polalta obtained 494 bp amplified product, and the susceptible control 1(326 was negative, indicating that the PCR amplification was specific. DNA was extracted from leaves of 1500 plants of BC6F1 backcross population for PCR verification of markers. The method of marker verification was the same as that in Example 3. Finally, two plants (No. 46 and No. 364) were detected as negative for NaChr344.2M marker but positive for NaChr3_64.6 M marker, and one plant (No. 59) was detected as positive for NaChr344.2M marker but negative for NaChr3_64.6 M marker. By TSWD resistance identification (the method was the same as that in Example 1), we found that plant No. 59 was susceptible to TSWD as it did not show HR after inoculation with avirulence gene, meanwhile plants No. 46 and No. 364 showed HR after inoculation with avirulence gene. The results showed No. 46 and No. 364 plants were TSWD resistance plants, and they were preliminarily determined as plants with effective shortened resistance introgressed segment. In order to clarify the shortened size of the resistant introgressed segment in tobacco plants No. 46 and No. 364, more molecular markers were developed (Table 5).

TABLE-US-00005 TABLE5 Molecularmarkersofresistance introgressedsegment(2) Markername Primername Primersequence NaChr3_54M NaChr3_54MF GTGGAGGATACGA TTACGCCTGTC (SEQIDNo.14) NaChr3_54MR GCCATCATTAAC TGCTTGACCAAC C (SEQIDNo.15) NaChr3_57M NaChr3_57MF GGGTGTGTTTCG GGTTGTGAATCC (SEQIDNo.16) NaChr3_57MR CAGTTTCCGCTT CCACGGTTTGA (SEQIDNo.17) NaChr3_58M NaChr3_58MF GCACGCCGTCCA CTTTGAATG (SEQIDNo.18) NaChr3_58MR ACGAGGCTAGAC AGGACCTACAA (SEQIDNo.19) NaChr3_59M NaChr3_59MF GCATTGTTCCGA CTTGTAGAATCC TT (SEQIDNo.20) NaChr3_59MR GTGCCAATAGTTA CCACTGTTCCAA (SEQIDNo.21)

[0149] The size of the resistance introgressed segments carried by the plants No. 59, No. 46 and No. 364 were detected using these molecular markers, and the results were shown in Table 6.

TABLE-US-00006 TABLE 6 Test results of markers in recombination plants Individual Marker name Resistance plant NaChr3_44.2M NaChr3_54M NaChr3_57M NaChr3_58M NaChr3_59M NaChr3_64.6M NaChr3_65.7M identification 59# Pos Neg Neg Neg Neg Neg Neg No HR 46# Neg Neg Neg Neg Pos Pos Neg Has HR 364# Neg Neg Neg Pos Pos Pos Neg Has HR N. alata Pos Pos Pos Pos Pos Pos Pos Has HR Polalta Pos Pos Pos Pos Pos Pos Pos Has HR K326 Neg Neg Neg Neg Neg Neg Neg No HR Note: 59#, 46# and 364# in the table represent plant No. 59, plant No. 46 and plant No. 364 respectively. Pos means positive, Neg means negative, and HR means hypersensitive reaction. Has HR means having TSWD resistance, and No HR means not having TSWD resistance.

[0150] From Table 6, we found that plant No. 46 contains a shorter resistance introgressed segment. NaChr344.2M, NaChr3_54M, NaChr3_57M, and NaChr3_58M are negative in plant No. 46. It means the introgressed segment has been shortened by more than 14 Mb in plant No. 46. By resistance identification, it was speculated that the resistance gene locus should locate between 58 Mb and 64.8 Mb on Chr3 of the N alata genome.

[0151] The molecular markers (NaChr4_2M and NaChr4_8M) located at both ends of the first deformity locus (DEF1) introgressed segment were used to detect plant No. 46 and the result was positive. Since plant No. 46 carried the DEF1 locus, it still had a severe deformed phenotype. Our results indicated that the DEF1 locus and RTSW (DEF2) locus were located on different chromosomes and should be segregated separately. Therefore, we tried to screen plants carrying only the RTSW (DEF2) introgressed segment among the progenies of plant No. 46. 248 plants of F2 self-crossing segregating population of plant No. 46 were subjected to screening, and a total of 60 plants carrying only the RTSW (DEF2) locus but no DEF1 locus were obtained. However, all of these 60 plants still showed severe deformed phenotypes. Among them, 34 plants were unable to bloom at all, and other 26 plants were delayed in flowering and few-flowered. Among them, 4 plants had only 1-2 flowers with low seed setting rate and seed quantity.

Example 7. Genetic Relationship of Linkage Drag Loci

[0152] According to the analysis of the disease resistance locus (RTSW) and the deformity loci (DEF), the genetic relationship between the RTSW locus and DEF loci was preliminarily revealed. We found that the deformed phenotype was determined by two loci. DEF1 was derived from 1-8.6 Mb on chromosome 4 of the genome of the wild tobacco N alata. The other locus (DEF2) was tightly linked to the resistance locus (RTSW) and located on the same genome region, 43.7 Mb to 64.8 Mb, of N alata Chr3. In the continuous backcrossing process of multiple generations, the DEF1 locus and RTSW (DEF2) locus showed co-segregation all the time. Through investigation of the F2 segregated population of plant No. 46, we speculate that the DEF1 locus is essential for the surviving of RTSW (DEF2) locus. If there is only RTSW (DEF2) locus, plants will show more severe deformity and poor fertility. If DEF1 and RTSW (DEF2) loci co-exist, plants will show severe deformed phenotypes, but the fertility is not significantly different from that of the parents Polalta and 1(326. Therefore, we proposed the following model: there may be a toxin gene (Toxin) near the RTSW (DEF2) locus, which alone may cause a more severe deformed phenotype and sterility, while there may be a detoxin gene (Detoxin) at the DEF1 locus. The RTSW (DEF2) locus can be stably inherited only in the presence of the DEF1 locus (FIG. 4).

Example 8. Simultaneous Removal of Two Linkage Drag Loci by Screening from a Super Large Population

[0153] According to our model, there may be a toxin gene near the RTSW (DEF2) locus, which alone may cause a more severe deformed phenotype and sterility, while there may be a detoxin gene at the DEF1 locus, and the RTSW (DEF2) locus can be inherited stably only in the presence of the DEF1 locus. Therefore, in order to segregate the DEF1 locus, it is necessary to segregate the gene segment that causes sterility at the RTSW (DEF2) locus simultaneously. In order to obtain a completely normal plant, the DEF2 locus must also be segregated. However, the introgressed segments of DEF1 and RTSW (DEF2) derived from wild tobacco N alata have low homology with the sequence in cultivated tobacco, so recombination inhibition will occur, resulting in a low recombination rate. In order to shorten the introgressed segments of DEF1 and RTSW (DEF2) simultaneously and obtain completely normal resistant plants, it is necessary to screen a huge segregation population. Deformed phenotypes can be used as the visible marker for screening at the seedling stage. For phenotypic screening, the BC7F1 backcross population was produced by using plant No. 46 as the male parent (RTSW(DEF2)/rtsw(def2), DEF1/def1 genotype) and 1(326 as the female parent, and were seeded in the seedling pond, with 1600 floating plates, about 100 seeds per plate. A total of more than 160,000 seedlings were subjected to screening. During the seedling stage, for deformed phenotypes, a total of 5 rounds of screening were carried out. Once the deformed seedlings were observed, they would be discarded during the screening. After critical screening, 12,000 completely normal seedlings were finally obtained for the next molecular marker screening.

[0154] Genomic DNA was extracted from the 12,000 tobacco plants for marker screening. First, molecular markers NaChr3_59M and NaChr3_64.6M located at both ends of the resistance introgressed segment of plant No. 46 were simultaneously used for screening. Using our optimized molecular marker screening system, 18 plants with at least one of the markers (NaChr3_59M and NaChr3_64.6M) positive were obtained (FIG. 5).

[0155] Next, the 18 plants were further tested using the markers at the DEF1 locus (NaChr4_2M and NaChr4_8M). All the plants were negative for primer pairs NaChr4_2MF/NaChr4_2MR and NaChr4_8MF/NaChr4_8MR, indicating that all the plants do not contain the DEF1 locus.

[0156] These 18 plants were then submitted to identify the resistance property to TSWD by using avirulence gene mediated HR (described in Example 1). By transiently expressing the avirulent gene NSm and observing the HR response, it can be determined whether there is a resistance introgressed segment. Finally, 5 plants that could be induced HR response when the avirulent gene was expressed were screened out of the 18 plants. The 5 plants have TSWD resistance, named as plant No. 1, plant No. 4, plant No. 11, plant No. 12, and plant No. 17.

[0157] In order to detect the size of the resistance introgressed segment carried by the 5 plants and select the shortest resistance introgressed segment for future breeding program, more molecular marker detection on the 5 selected resistant plants were carried out. Using the sequence of the genome, two new pairs of molecular marker primers were further developed for detection based on the resistance introgressed segment (Table 7).

TABLE-US-00007 TABLE7 Molecularmarkersoftheresistance introgressedsegment(3) Primer Marker Primer sequence name name (5'-3') NaChr3_ NaChr3_ TCTTACCTCTCCT 60M 60MF ACTACTCCTCCAT C (SEQIDNo.22) NaChr3_ GCATCTTCTTCTTC 60MR TCCGTATCTCCAA (SEQIDNo.23) NaChr3_ NaChr3_ CTTAGCAGGCAACC 62.6M 62.6MF AGACAG (SEQIDNo.24) NaChr3_ GAGAATGAGCAAGA 62.6MR GAATGTGTTAG (SEQIDNo.25)

[0158] It was found that plant No. 12 carried the shortest resistance introgressed segment. Plant No. 12 was only positive for NaChr3_59M marker, but negative for other markers (Table 8).

TABLE-US-00008 TABLE 8 Marker detection results of recombination plants Marker name Resistance Plant NaChr3_59M NaChr3_60M NaChr3_62.6M NaChr3_64.6M identification Phenotype 1# Pos Pos Pos Neg Has HR Normal 4# Pos Pos Neg Neg Has HR Normal 11# Pos Pos Pos Neg Has HR Normal 12# Pos Neg Neg Neg Has HR Normal 17# Pos Pos Pos Neg Has HR Normal BC6F1-46# Pos Pos Pos Pos Has HR Deformed N. alata Pos Pos Pos Pos Has HR Not defined Polalta Pos Pos Pos Pos Has HR Deformed K326 Neg Neg Neg Neg No HR Normal Note: 1#, 4#, 11#, 12# and 17# in the table represent plant No. 1, plant No. 4, plant No. 11, plant No. 12, plant No. 17 respectively. Pos means positive, Neg means negative, and HR means hypersensitive reaction. Has HR means having TSWD resistance, and No HR means not having TSWD resistance.

Example 9. Phenotype and TSWD Resistance Test of Self-Crossed Progenies of the Plant without linkage drag

[0159] Whether the phenotype is completely restored to normal is the key point of the invention. Therefore, the phenotypes of the 5 resistant plants (No. 1, No. 4, No. 11, No. 12, and No. 17) selected in Example 8 were observed at seedling stage and adult stage. The observation results showed that the phenotypes of the 5 resistant plants at seedling stage and adult stage had no visible linkage drag, the morphology improved significantly, and the main agronomic traits such as plant height, leaves and flowering period were no significant different with the cultivar K326 (FIG. 6).

[0160] In order to detect whether the resistance introgressed segment still confers complete resistance to TSWV, self-crossed progenies of the plant No. 12 were inoculated with TSWV. The self-crossed progenies of the plant No. 12 were genotyped using molecular marker detection primers NaChr3_59MF/NaChr3_59MR. The plants that were positive or negative for the molecular marker were selected for rubbing inoculation with TSWV, and the symptoms were observed at two weeks postinoculation. Uninoculated systemic leaves at the top of the plants were collected for virus detection by ELISA. The method was as follows: (1) took 1-2 g TSWV source, put it in a mortar, added 5-10 mL of TSWV inoculation buffer (0.1M pH 7.0 phosphate buffer sterilized at 121 C. for 20 minutes; Within half an hour before inoculation, added 0.2 g sodium sulfite and 10 uL beta-mercaptoethanol per 100 mL phosphate buffer to make TSWV inoculation buffer and placed it on ice) and 2-3 g of carborundum (200-400 mesh), fully ground on ice until they were evenly mixed to obtain TSWV inoculum. For mechanical inoculation, carborundum (200-400 mesh) was first evenly sprinkled on the surface of a tobacco leaf to be inoculated at an amount of 0.1-0.2 g per leaf, then held the tobacco leaf to be inoculated with one hand, and rubbed the TSWV sap gently and evenly with the other hand from the base to the tip of the tobacco leaf to be inoculated; the dosage of TSWV inoculum was 50-100 ul per leaf; (2) after rubbing, rinsed the inoculated leaves with water, then cultivated the plants in the dark under a temperature of 22-25 C. with humidity of 60-80% for one day, and then moved the plants to growth chamber with a temperature of 22-25 C., a photoperiod of 14h day/10h night, and a humidity of 80%; (3) from the 9th day after inoculation, took fresh tobacco leaves every 7 days and used the double antibody sandwich ELISA method (Agdia, Cat. No. SRA 39300/0096) to detect TSWV and calculated the incidence of TSWD a; a total of four tests were performed. In order to reduce the error of human operation, for ELISA test, each sample was repeated three times. The operation steps of the double antibody sandwich ELISA method were carried out according to the instructions (Agdia, Cat. No. SRA 39300/0096).

[0161] The total number of infected plants was the total number of positive plants in the four tests. Incidence a=(number of susceptible plants of the variety/total number of plants of the variety)100%. The results of genotyping, symptom observation after inoculation and ELISA test showed that 12 plants carrying the resistance introgressed segment (detected as positive for NaChr3_59M marker) were completely resistant to the disease, while 12 plants without the resistance introgressed segment (detected as negative for NaChr3_59M marker) were 100% susceptible (FIG. 7).

Example 10. Analysis of Agronomic Traits of Materials without Linkage Drag

[0162] The plant No. 12 carrying the shortest RTSW introgressed segment was self-crossed, and the self-crossed progenies were genotyped using molecular marker detection primers NaChr3_59MF/NaChr3_59MR. 1000 homozygous plants (genotype RTSW/RTSW) of F3 generation were growth in the field in 2021 (indicated as K326-RTSW in Table 9), and the cultivar 1(326 was growth as a control. There was no difference between all K326-RTSW plants (carrying the shortest RTSW introgressed segment) and the control plants (cultivar K326) in terms of germination rate, survival rate, and field growth at the seedling stage. At the vegetative and reproductive stage, 10 plants were randomly selected to measure plant height, stem circumference, leaf number, leaf length, leaf width and other performances to test the agronomic traits of K326-RTSW (Table 9).

TABLE-US-00009 TABLE 9 Analysis of agronomic traits Natural Natural Total plant plant number Stem height height of leaves circumference at full at green Topping at full at full flowering fruit plant flowering flowering stage (cm) stage (cm) height (cm) stage (blade) stage (cm) K326-RTSW 133.45 12.08 153.9 13.46 134.86 10.35 26.8 2.48 10 0.77 K326 139.2 18.61 159.2 18.93 141.95 8.08 26.4 1.28 10.23 0.83 p-value 0.4397 0.1022 0.1741 0.3104 0.3329 Midstalk Midstalk Midstalk Midstalk leaf leaf leaf leaf Stem length width length width at circumference at full at full at green green at green fruit flowering flowering fruit fruit stage (cm) stage (cm) stage (cm) stage (cm) stage (cm) K326-RTSW 10.69 0.79 71.96 3.46 32.44 2.89 73.54 4.52 32.41 3.06 K326 10.51 0.9 68.83 4.74 34.5 3.sup. 70.26 5.62 32.54 2.66 p-value 0.2193 0.0918 0.2003 0.5050 0.7599

[0163] From the table, we can find that the linkage drag has been completely segregated from the short RTSW introgressed segment. The obtained K326-RTSW variety carrying the short RTSW introgressed segment had no significant difference from the elite cultivar K326 in terms of plant height, stem circumference, number of leaves, leaf length, leaf width and other agronomic traits.