SSR markers linked to the resistance gene wBS1_t of tobacco black shank race 1 and their applications

Abstract

This invention uses wild-type tobacco *Nicotiana stocktonii*, which is highly resistant to black shank disease race 1, as the starting material. Two co-dominant SSR markers closely linked to the *Nicotiana stocktonii* resistance gene *wBS1_t* were obtained and used to detect the presence of the *Nicotiana stocktonii* resistance gene *wBS1_t* in the tobacco genomic DNA. The SSR markers provided by this invention are rapid, stable, accurate, and low-cost, and can be used as molecular marker-assisted selection for the *wBS1_t* gene in breeding for resistance to *Nicotiana stocktonii* race 1.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an SSR marker linked to the resistance gene wBS1_t of tobacco black shank race 1 and its application. Background Technology

[0002] Tobacco black shank is one of the most serious diseases affecting tobacco production worldwide. Currently, four physiological races of the fungus responsible for tobacco black shank have been reported. Race 0 is the dominant physiological race infecting tobacco; races 2 and 3 are not yet widespread. With in-depth research into race 0 of black shank and the widespread application of tobacco varieties resistant to race 0, there is a significant risk that the dominant race of the fungus in tobacco-growing soil may shift from race 0 to race 1.

[0003] Currently, the main sources of resistance to black shank disease (SCR) in tobacco production are concentrated in the cigar tobacco resources Beinhart 1000 and Florida 301. Vontimitta et al. conducted QTL analysis on black shank resistance in Beinhart 1000 and detected 6 QTLs associated with black shank resistance. The major QTL locus with the largest contribution rate was located in linkage group 8 (Vontimitta V, Lewis R S. Mapping of quantitative trait loci affecting resistance to Phytophthora nicotianae in tobacco (Nicotiana tabacum L.) line Beinhart-1000. Mol Breeding, 2012, 29:89-98.). Zhang et al. also located the QTL with the largest effect (qBS7) in Beinhart 1000-1. Ma et al. further analyzed the location and effect of this QTL (named Phn7.1), increased the marker density by developing SNPs, and narrowed the mapping interval to about 3 cM using a series of near-isogenic lines (NILs). This can be used for molecular marker-assisted selection breeding and candidate gene cloning (Zhang Y, Guo X, Yan X, Ren M, Jiang C, Cheng Y, Wen L, Liu D, Zhang Y, Sun M, Feng Q, Yang A, Chenga L. Identification of stably expressed QTL for resistance to black shank disease in tobacco (Nicotianatabacum L.) line Beinhart 1000-1. The Crop J., 2018, 6:282-290; Ma JM, Heim C, Humphry M, Nifong JM, Lewis R S. Genetic analysis of Phn7.1, a major QTL conferring partial resistance to Phytophthora nicotianae in Nicotianatabacum. Mol Breeding, 2019, 39:11.).Xiao et al. used a combination of natural disease development in the field and artificial inoculation in a greenhouse to conduct QTL analysis on the black shank resistance of Florida 301 cigar tobacco. The results showed that two major resistance QTLs were obtained (Bingguang Xiao, Katherine Drake, Vijay Vontimitta, Zhijun Tong, Xueting Zhang, Meiyun Li, Xiaodong Leng, Yongping Li and Ramsey S. Lewis. Location of genomic regions contributing to Phytophthora nicotianae resistance in tobacco cultivar Florida 301. Crop Sci., 2013, 53: 473-481.).

[0004] Chinese patent application 202111361796.X (A codominant SSR marker closely linked to the Beinhart1000-1 black shank resistance gene qBS01 in cigars and its application) is a marker developed from a cigar resistance source and a quantitative trait of tobacco black shank race 1 resistance gene / QTL.

[0005] Chinese Patent ZL 2017 1 1124725.1 (An SSR marker linked to the resistance gene Bs_t for races 0 and 1 of tobacco black shank and its application) provides the gene Bs_t, and the resistance to race 1 of black shank originates from yellow tobacco.

[0006] To date, no molecular markers have been reported for the development of resistance genes wBS1_t for black shank race 1, which is a quality trait and has a wild tobacco origin. Summary of the Invention

[0007] The first objective of this invention is to provide a novel resistance source for black shank disease race 1 derived from wild tobacco, wherein the resistance gene of this resistance source is wBS1_t; the second objective is to provide a codominant SSR marker linked to this resistance gene wBS1_t, used to detect the presence of the tobacco black shank disease race 1 resistance gene wBS1_t in tobacco genomic DNA.

[0008] The first objective of this invention is achieved as follows: the black shank resistance gene wBS1_t derived from wild tobacco is introduced into the newly obtained tobacco breeding material 87St after chromosome doubling by using a distant cross between wild tobacco Nicotiana stocktonii, which is highly resistant to black shank race 1, and haploid induction.

[0009] The second objective of this invention is achieved as follows: the codominant SSR markers linked to the resistance gene wBS1_t of tobacco black shank disease race 1 are numbered NtP20445 and NtP16198, and the nucleotide sequences of their PCR amplification products are shown in SEQ ID No. 1 and SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0010] To facilitate a simple, precise, and efficient selection of varieties resistant to tobacco black shank race 1, and to specifically select progeny materials derived from wild tobacco containing the black shank resistance gene wBS1_t, this invention has conducted the following specific research:

[0011] 1. Using wild tobacco Nicotiana stocktonii, which is highly resistant to black shank disease race 1, as the starting material, through distant hybridization with bridge tobacco germplasm, haploid induction, and chromosome doubling, breeding material 87St, which contains the resistance gene wBS1_t derived from wild tobacco and is highly resistant to black shank disease race 1, was obtained.

[0012] 2. Using the above-mentioned disease-resistant material 87St and the susceptible variety Yunyan 87 as parents, a second-generation (F2) genetic population containing 300 plants was constructed through hybridization and bagging self-pollination.

[0013] 3. Using bulked segregation analysis (BSA) and single-grain black shank cultivar trellis inoculation, two codominant SSR markers linked to the resistance gene wBS1_t of tobacco black shank race 1 were screened. These markers can be used for marker-assisted selection of the black shank resistance gene wBS1_t, thereby achieving precise and efficient utilization of molecular marker-assisted selection (MAS) in the breeding of new lines resistant to tobacco black shank race 1.

[0014] The following numbers NtP20445 and NtP16198 are the numbers of two codominant SSR markers linked to the resistance gene wBS1_t of tobacco black shank race 1 provided by this invention.

[0015] The nucleotide sequences of the PCR amplification products of NtP20445 are: SEQ ID No. 1 and SEQ ID No. 2;

[0016] The nucleotide sequences of the PCR amplification products of NtP16198 are: SEQ ID No. 3 and SEQ ID No. 4.

[0017] The primer sequences for the two sites corresponding to the two SSR markers are as follows:

[0018] The primer sequence for NtP20445 is as follows:

[0019] SEQ ID No.5: 5'-CAAGAACAATGTGTCAACTGTGAA-3',

[0020] SEQ ID No.6: 5'-ATGGCAAAATGTTGCATCTC-3';

[0021] The primer sequence for NtP16198 is:

[0022] SEQ ID No.7: 5'-TCCAGAATGCATTATCAGAGTTTC-3',

[0023] SEQ ID No. 8: 5'-ACAAGGAGGAATGCGAACAG-3'.

[0024] The molecular marker provided by this invention for detecting the black shank resistance gene wBS1_t of race 1, which is a resistance source from wild tobacco Nicotiana stocktonii, has the following significant advantages compared with existing technologies (black shank resistance gene markers reported in patent and non-patent literature):

[0025] (1) Different types of antigens

[0026] The existing resistance sources are derived from cultivated tobacco varieties—cigar varieties (Beinhart 1000-1 and Florida 301) and yellow-flowered tobacco (N. rustica); while the resistance source for black shank disease path 1 involved in this invention is derived from a non-cultivated tobacco species—wild tobacco (Nicotiana stocktonii).

[0027] (2) Different physiological races of resistance

[0028] The existing technology involves resistance to black shank disease against race 0; while the black shank disease resistance involved in this invention is resistance against race 1.

[0029] (3) Different resistance levels

[0030] Although patent application 202111361796.X targets resistance to physiological race 1, its resistance is a horizontal resistance of quantitative traits; while the black shank resistance involved in this invention is a vertical resistance of qualitative traits, which is strong and durable.

[0031] (4) There are no adverse chain reactions.

[0032] Existing black shank resistance genes all suffer from linkage slack, which is an unfavorable trait in tobacco production. However, the black shank resistance gene wBS1_t involved in this invention does not have unfavorable linkage slack. Therefore, it is beneficial for the large-scale promotion and application of varieties containing the wBS1_t gene that are resistant to black shank race 1.

[0033] The two codominant SSR markers are designated NtP20445 and NtP16198. Their relationship is as follows: these two markers allow for precise, clear, rapid, and efficient localization of the target gene (the resistance gene wBS1_t for tobacco black shank race 1). In other words, the target gene is "locked" between these two markers (see appendix). Figure 3 Directly detecting the target trait (resistance to tobacco black shank race 1) is extremely time-consuming, labor-intensive, costly, and requires a high level of knowledge of environmental and plant pathology conditions. However, when using the two markers mentioned above to perform PCR amplification detection on the genome of tobacco plants at any stage, it is only necessary to look at the sequences of the products amplified by the two markers to determine the resistance to black shank race 1 of the tested plant.

[0034] Explanation of the technology of this invention

[0035] When used as a detection reagent, both markers must be used simultaneously. Only by using both markers together can the presence of the target gene (wBS1_t) located between the two markers be accurately and clearly determined. If only one marker is used, the presence of the target gene adjacent to that marker cannot be accurately and clearly determined. That is, if only the PCR amplification product sequence information of one of the two markers is known, the presence of the target gene linked to that marker cannot be accurately determined (the target gene may or may not be present at this time).

[0036] Each marker corresponds to two sequences (PCR amplification products consist of two sequences).

[0037] Because of the disease resistance of organisms, there are always resistant individuals (varieties) and susceptible individuals (varieties). Regarding disease resistance (the target trait), there are two traits: resistance and susceptibility. Since traits (phenotypes) are determined by internal genes, there are two types of genes (can also be understood as alleles): resistance and susceptibility. Similarly, as a marker closely linked to the target gene (wBS1_t), its PCR amplification product sequence will be different in resistant and susceptible plants (i.e., different PCR amplification product sequences in resistant and susceptible plants correspond to resistance and susceptibility sequences, respectively; if the PCR amplification sequence of the marker is the same in resistant and susceptible plants, i.e., only one sequence, it indicates that the marker is not linked to the target gene). In short, because plants have two different genes for resistance and susceptibility, the PCR amplification product sequence of a marker linked to that gene will also be different in resistant and susceptible plants (i.e., two different sequences are amplified, one for resistance and one for susceptibility).

[0038] The relationship between the two sequences is as follows: the PCR amplification product of a linkage marker (such as NtP20445) in a plant will produce two different sequences, which are determined by the resistance (resistant or susceptible) of the plant being tested: if the plant is resistant, its PCR amplification product sequence length is 189 bp (SEQ ID No. 1); if the plant is susceptible, its PCR amplification sequence length is 210 bp (SEQ ID No. 2). In other words, the relationship between the two PCR amplification product sequences of a marker is the resistance / susceptibility relationship of the plant being tested.

[0039] Both sequences must be included in a single tag.

[0040] Since plant disease resistance is determined by the presence of disease resistance genes within its genome, when the resistance gene cannot be directly located, its presence can only be determined by molecular markers tightly linked to the target gene (the resistance gene wBS1_t in this invention). To accurately, quickly, efficiently, and clearly determine plant resistance (resistance or susceptibility) using markers tightly linked to the target gene, both markers must be used simultaneously. Only when both markers are used simultaneously can the presence of the target gene located in the middle be accurately and clearly determined, thus clearly indicating the phenotype (resistance or susceptibility) of the tested plant. Specifically, in this invention:

[0041] The codominant SSR markers linked to the resistance gene wBS1_t in tobacco black shank race 1 are designated NtP20445 and NtP16198. Their PCR amplification products have the following nucleotide sequences: SEQ ID No. 1 (anti-) and SEQ ID No. 2 (susceptible), and SEQ ID No. 3 (anti-) and SEQ ID No. 4 (susceptible), respectively. (That is, each marker can amplify both anti- and susceptible sequences; NtP20445 amplifies SEQ ID No. 1 (anti-) and SEQ ID No. 2 (susceptible), and NtP16198 amplifies SEQ ID No. 3 (anti-) and SEQ ID No. 4 (susceptible)).

[0042] The genomic DNA of the tobacco plant to be tested was amplified using primers with the NtP20445 and NtP16198 sequences, respectively. The PCR amplification products were then analyzed. If the PCR amplification products contained sequences as shown in SEQ ID No. 1 and SEQ ID No. 3, it indicated that the tested tobacco plant contained a homozygous allele for resistance to black shank race 1, denoted as wBS1_t (resistant, and homozygous resistance, meaning the resistance will not segregate in offspring (the resistance remains unchanged)). If the PCR amplification products contained sequences as shown in SEQ ID No. 2 and SEQ ID No. 4, it indicated that the tested tobacco plant did not contain a homozygous allele for resistance to black shank race 1, denoted as wbs1_t (susceptible, and homozygous susceptible, meaning the susceptibility will not segregate in offspring (the susceptibility remains unchanged)). If the PCR amplification products contained sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, or contained sequences as shown in SEQ ID No. 4, it indicated that the tested tobacco plant did not contain a homozygous allele for resistance to black shank race 1, denoted as wbs1_t (susceptible, and homozygous susceptible, meaning the susceptibility will not segregate in offspring (the susceptibility remains unchanged)). The sequence shown in No. 3 and SEQ ID No. 4, or the sequence shown in SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 4, or the sequence shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, indicates the presence of a heterozygous allele for the resistance to black shank race 1, with the genotype wBS1_t wbs1_t (resistance, but heterozygous resistance, meaning that the offspring will exhibit segregation between resistant and susceptible individuals (the offspring will not retain the resistance indefinitely)).

[0043] Sequence information:

[0044] SEQ ID No. 1 and SEQ ID No. 2 are amplified sequences of the codominant SSR marker numbered NtP20445;

[0045] SEQ ID No. 3 and SEQ ID No. 4 are amplified sequences of the codominant SSR marker numbered NtP16198;

[0046] SEQ ID No. 5 and SEQ ID No. 6 are primer sequences for the codominant SSR marker numbered NtP20445;

[0047] SEQ ID No. 3 and SEQ ID No. 4 are primer sequences for the codominant SSR marker numbered NtP16198. Attached Figure Description

[0048] Figure 1 This is a gel electrophoresis image of the PCR amplification products of the codominant SSR marker NtP20445, which is linked to the resistance gene wBS1_t derived from wild tobacco black shank race 1, in five materials.

[0049] Among them, the codominant SSR marker is numbered NtP20445; P1 is the parent with the wBS1_t resistance gene derived from wild tobacco black shank race 1 (breeding material-87St); P2 is the susceptible parent (flue-cured tobacco variety-Yunyan 87); F1 is the first generation of hybrids between the two parents; B1 is a mixed DNA pool constructed from individuals in the second generation (F2) population with resistance to black shank race 1 (containing the wBS1_t gene); B2 is a mixed DNA pool constructed from individuals in the second generation (F2) population with the disease (not containing the wBS1_t gene); M is a 500bp DNA ladder with fragments of length from top to bottom as follows: 500bp, 400bp, 300bp, 200bp, and 100bp.

[0050] Figure 2 This is a gel electrophoresis image of the PCR amplification products of the codominant SSR marker NtP16198, which is linked to the resistance gene wBS1_t derived from wild tobacco black shank race 1, in five materials.

[0051] Among them, the codominant SSR marker is numbered NtP16198; P1 is the parent with the wBS1_t resistance gene derived from wild tobacco black shank race 1 (breeding material-87St); P2 is the susceptible parent (flue-cured tobacco variety-Yunyan 87); F1 is the first generation of hybrids between the two parents; B1 is a mixed DNA pool constructed from individuals in the second generation (F2) population with resistance to black shank race 1 (containing the wBS1_t gene); B2 is a mixed DNA pool constructed from individuals in the second generation (F2) population with the disease (not containing the wBS1_t gene); M is a 500bp DNA ladder with fragments of length from top to bottom as follows: 500bp, 400bp, 300bp, 200bp, and 100bp.

[0052] Figure 3The codominant SSR markers NtP20445 and NtP16198 are linked to the resistance gene wBS1_t of the first physiological race of tobacco black shank, respectively.

[0053] The left side shows the genetic distance (in cM) between the SSR marker and the resistance gene wBS1_t of tobacco black shank disease race 1; the right side shows the name of the SSR marker and the name of the resistance gene wBS1_t. Detailed Implementation

[0054] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the relevant product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased.

[0055] The application of the codominant SSR marker linked to the resistance gene wBS1_t of the first physiological race of tobacco black shank, as described in this invention, is the application of the codominant SSR marker linked to the resistance gene wBS1_t of the first physiological race of tobacco black shank in detecting the presence of the resistance gene wBS1_t of the first physiological race of tobacco black shank in tobacco genomic DNA.

[0056] The application of the codominant SSR marker linked to the resistance gene wBS1_t for tobacco black shank disease race 1 involves amplifying the genomic DNA of the tobacco plant to be tested using primers with the NtP20445 and NtP16198 sequences, respectively. The PCR amplification products are then detected. If the PCR amplification products contain sequences as shown in SEQ ID No. 1 and SEQ ID No. 3, the tobacco plant possesses the homozygous allele wBS1_t for resistance to black shank disease race 1. If the PCR amplification products contain sequences as shown in SEQ ID No. 2 and SEQ ID No. 4, the tobacco plant does not possess the homozygous allele wbs1_t for resistance to black shank disease race 1. If the PCR amplification products contain sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, or sequences as shown in SEQ ID No. 3 and SEQ ID No. 4, or sequences as shown in SEQ ID No. 1, SEQ ID No. 3, and SEQ ID No. 4, or sequences as shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 1, SEQ ID No. 1, SEQ ID No. 3, and SEQ ID No. 4, or sequences as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, or sequences as shown in ...2, SEQ ID No. 3, and SEQ ID No. 4, or sequences as shown in The sequences shown in No. 3, SEQ ID No. 1, and SEQ ID No. 2 are the heterozygous genes wBS1_t and wbs1_t that contain resistance to black shank race 1 in the tested tobacco plants.

[0057] The invention will be further illustrated below with specific implementation examples:

[0058] Example 1 uses the Biosegregation-Group Analysis (BSA) method combined with the single-grain black shank vine wound inoculation method to screen for codominant SSR markers linked to the resistance gene wBS1_t of tobacco black shank race 1.

[0059] I. Experimental Materials

[0060] Using Yunyan 87, a flue-cured tobacco variety susceptible to black shank disease No. 1, as the female parent and 87St, a breeding material containing the wBS1_t gene and highly resistant to black shank disease No. 1, as the male parent, 300 plants were hybridized and self-pollinated to obtain the F2 generation of self-pollinated plants as the genetic population.

[0061] II. Obtaining Phenotypic Data on Blackleg Resistance of Race 1 in Parental Lines and F2 Population

[0062] The single-grain inoculation method using *Blackshankia gravidarum* was employed. Parental and F2 populations were artificially inoculated indoors. Disease incidence was observed 3 days post-inoculation. Following the national standard GB / T 23222-2008 (Grading and Survey Methods for Tobacco Diseases and Pests), three surveys were conducted on days 7, 14, and 21 post-inoculation, and the Disease Index (DI) was calculated. The DI values ​​of resistance to race 1 of blackshankia obtained from 300 F2 individuals were used as phenotypic data for subsequent BSA analysis.

[0063] III. SSR Marker Analysis

[0064] Tobacco genomic DNA extraction: Either the conventional CTAB method or a plant tissue DNA extraction kit can be used. For the method, please refer to existing literature or the instructions in the kit.

[0065] PCR amplification and electrophoresis detection: The PCR amplification system is a conventional system and can be referred to in published literature. The labeling annealing temperature provided in this invention is 60℃. The PCR amplification program information can be referred to in relevant literature. Electrophoresis detection also adopts conventional methods and can be referred to in relevant published literature.

[0066] IV. Construction of disease-resistant (including wBS1_t gene) and disease-susceptible (excluding wBS1_t gene) genome pools

[0067] Genomic DNA was collected from 15 extremely resistant and 15 susceptible plants from the F2 segregating population using the Biosegregating Segregating Population (BSA) method. These were then mixed in equal amounts to construct resistant and susceptible genomic DNA pools for primer polymorphism screening and marker linkage analysis. During the seedling stage, following the method described in Example 1, PCR amplification was performed on five materials (P1 (87St), P2 (Yunyan 87), F1 (first generation hybrid), B1 (resistant pool), and B2 (susceptible pool) using whole-genome SSR molecular markers to screen for molecular markers linked to the resistance gene wBS1_t of tobacco black shank disease race 1. The screening results are shown below. Figure 1 and Figure 2 As shown: the codominant SSR markers NtP20445 and NtP16198 are linked flanking the resistance gene wBS1_t of the first physiological race of tobacco black shank disease, respectively. The banding pattern of marker NtP20445 is completely consistent with that of the susceptible parent (P2-Yunyan 87) in the susceptible pool (B2), showing a specific band of 210 bp (sequence as shown in SEQ ID NO.2); the banding pattern is completely consistent with that of the F1 in the resistant pool (B1), showing two codominant specific bands (simultaneously showing specific bands of both resistant and susceptible parents, that is, simultaneously showing sequences as shown in SEQ ID NO.1 and SEQ ID NO.2); among them, the sequence shown in SEQ ID NO.1 is 189 bp in length, which is the specific PCR amplification band of the codominant SSR marker NtP20445 in the resistance gene wBS1_t derived from the first physiological race of tobacco black shank disease. Similarly, the banding pattern of marker NtP16198 was completely identical in the susceptible pool (B2) and the susceptible parent (P2-Yunyan 87), showing a specific band of 214 bp (sequence as shown in SEQ ID NO.4); the banding pattern was completely identical in the resistant pool (B1) and the F1, showing two co-dominant specific bands (simultaneously exhibiting specific bands of both resistant and susceptible parents, i.e., simultaneously showing sequences as shown in SEQ ID NO.3 and SEQ ID NO.4). Among them, the sequence shown in SEQ ID NO.3 is 278 bp in length and is a specific PCR amplification band of the co-dominant SSR marker NtP16198 derived from the resistance gene wBS1_t of wild tobacco black shank disease path 1.

[0068] The above results indicate that markers NtP20445 and NtP16198 are linked to the resistance gene wBS1_t of the first physiological race of tobacco black shank, respectively, and that the markers are co-dominant. The presence of only the sequences shown in SEQ ID NO.1 (189bp) and SEQ ID NO.3 (278bp) in the PCR amplification product indicates that the tested tobacco contains the homozygous wBS1_t gene and possesses resistance to black shank race 1; the presence of only the sequences shown in SEQ ID NO.2 (210bp) and SEQ ID NO.4 (214bp) in the PCR amplification product indicates that the tested tobacco contains the homozygous wbs1_t gene but does not possess resistance to black shank race 1; the presence of both sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or sequences shown in SEQ ID NO.3 and SEQ ID NO.4, or sequences shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4, or sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.4, indicates the presence of the homozygous wBS1_t gene but does not possess resistance to black shank race 1; the presence of both sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or sequences shown in SEQ ID NO.3 and SEQ ID NO.4, indicates the presence of the homozygous wBS1_t gene and does not possess resistance to black shank race 1. The sequence shown in No. 3 indicates that the tested tobacco contains the heterozygous wBS1_twbs1_t gene and possesses resistance to black shank race 1. See details. Figure 1 and Figure 2 .

[0069] Example 2: Validation and application of co-dominant linkage markers in F2 population individual plants

[0070] Using the codominant SSR markers NtP20445 and NtP16198, which are tightly linked to the resistance gene wBS1_t for tobacco black shank disease race 1, genotypic analysis was performed on individual plants of the F2 population (Yunyan 87×87St) at the seedling stage, obtaining genotypic data for each individual plant in the F2 population. On the other hand, the single-grain black shank disease spore inoculation method was used to inoculate and identify the black shank disease race 1 in each line, obtaining the resistance phenotype values ​​for each line. Finally, the genotypic data and black shank disease race 1 resistance phenotype values ​​of 300 F2 plants were analyzed, revealing that the genotypic values ​​and phenotype values ​​of the two codominant SSR markers NtP20445 and NtP16198 disclosed in this invention were completely consistent, i.e., the consistency rate reached 100%. The specific analytical methods are as follows: When the DI value of each strain of black shank disease 1 physiological race obtained by single-grain black shank disease inoculation method is lower than or equal to that of the resistant parent 87St, the genotype of this strain also shows the sequences shown in SEQ ID NO.1 (189bp) and SEQ ID NO.3 (278bp), which is the homozygous genotype wBS1_t for resistance to black shank disease 1 physiological race; when the DI value of each strain of black shank disease 1 physiological race obtained by identification is equal to or higher than that of the susceptible parent Yunyan 87, the genotype of this strain also shows the sequences shown in SEQ ID NO.2 (210bp) and SEQ ID NO.4 (214bp), which is the homozygous genotype wbs1_t for susceptibility to black shank disease 1 physiological race. wbs1_t; When the DI value of each identified strain of black shank disease 1 physiological race is between that of the susceptible parent Yunyan 87 and the resistant parent 87St, that is, similar to that of the F1 generation, the genotype of this strain also simultaneously presents the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or contains the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, or contains the sequences shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4, or contains the sequences shown in SEQ ID NO.3, SEQ ID NO.1 and SEQ ID NO.2, which is the heterozygous genotype wBS1_twbs1_t of the black shank disease resistant physiological race 1.

[0071] The above results indicate that the codominant markers NtP20445 and NtP16198 are linked to the resistance gene wBS1_t derived from wild tobacco black shank race 1, and these two markers are located flanking the target gene (wBS1_t). The genetic distances between markers NtP20445 and NtP16198 and the target gene wBS1_t are 1.96 cM and 1.02 cM, respectively. (See [link to relevant documentation]). Figure 3By utilizing the two co-dominant linked SSR markers mentioned above, we can accurately, efficiently, conveniently, and at low cost identify the resistance to Blackleg 1 physiological race at any growth stage of tobacco. Furthermore, we can clearly identify the genotype status of Blackleg 1 resistance in the tested plants. This not only improves the scientific rigor and predictability of breeding new flue-cured tobacco varieties resistant to Blackleg 1 physiological race, but also accelerates the breeding process.

[0072] sequence list

[0073] SEQ ID NO.1:

[0074] CAAGAACAATGTGTCAACTGTGAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGA

[0075] AGAAGAAGAAGAAGAAGAAGAAAAAGAAAGAAACGACATCAACAATTTGTCAACCAT

[0076] GAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAGATGCAGA

[0077] GATGCAACATTTTGCCAT

[0078] SEQ ID NO.2:

[0079] CAAGAACAATGTGTCAACTGTGAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGA

[0080] AGAAGAAGAAGAAGAAGAAGAAAAAGAAAGAAACGACATCAACAATTTGTCAACCAT

[0081] GAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAG

[0082] AAGAAGAAGAAGAGATGCAGAGATGCAACATTTTGCCAT

[0083] SEQ ID NO.3:

[0084] TCCAAGAATGCATTATCAGAGTTTCTTATATATATATATATATATATATATATATATATATATATATA

[0085] TATATATATATATATATATATATATATATATATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTTT

[0086] TCATGTATTTATGCACTGTTATAGTCTTTTTTCTCATACTTCATATTTCTCTTTGCTAATAGGA

[0087] TTACTCTTTGAGGGAGTATAACATGTTTAAAAATCAGGATTGGGATGTTTTGTTATAAATCT

[0088] GTTCGCATTCCTCCTTGT

[0089] SEQ ID NO.4:

[0090] TCCAGAATGCATTATCAGAGTTTCTTATATATATATATATATATATGTGTGTGTGTGTGTGTGT

[0091] GTGTTTTCATGTATTTATGCACTGTTATAGTCTTTTTTCTCATACTTCATATTTCTCTTTGCTA

[0092] ATAGGATTACTCTTTGAGGGAGTATAACATGTTTAAAAATCAGGATTGGGATGTTTTGTTAT

[0093] AAATCTGTTCGCATTCCTCCTTGT

[0094] SEQ ID NO.5:

[0095] CAAGAACAATGTGTCAACTGTGAASEQ ID NO.6:ATGGCAAAATGTTGCATCTCSEQ ID NO.7:TCCAGAATGCATTATCAGAGTTTCSEQ ID NO.8:ACAAGGAGGAATGCGAACAG

Claims

1. The application of a primer for detecting an SSR marker linked to a resistance gene in tobacco black shank race 1, characterized in that, The SSR markers are designated NtP20445 and NtP16198; the sequences of the NtP20445 PCR amplification products are SEQ ID NO:1 and SEQ ID NO:2; the sequences of the NtP16198 PCR amplification products are SEQ ID NO:3 and SEQ ID NO:4; the sequences of the NtP20445 PCR amplification primers are SEQ ID NO:5 and SEQ ID NO:6; and the sequences of the NtP16198 PCR amplification primers are SEQ ID NO:7 and SEQ ID NO:

8. The application is to detect whether tobacco contains the resistance gene for black shank race 1. If the PCR amplification products simultaneously contain sequences as shown in SEQ ID No. 1 and SEQ ID No. 3, it indicates that the tested tobacco plant contains a homozygous allele for resistance to black shank race 1, and the genotype is denoted as wBS1_t. If the PCR amplification product contains both sequences as shown in SEQ ID No. 2 and SEQ ID No. 4, it indicates that the tested tobacco plant does not contain the homozygous allele of the anti-black shank race 1, and the genotype is recorded as wbs1_t wbs1_t; If the PCR amplification product contains the sequences shown in SEQ ID No. 1 and SEQ ID No. 2, or the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, or the sequences shown in SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 4, or the sequences shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, it indicates that the tested tobacco plant contains a heterozygous allele of the physiological race 1 for resistance to black shank disease, and the genotype is recorded as wBS1_t wbs1_t.

Images