SNP (Single Nucleotide Polymorphism) site marker in tobacco genome, KASP primer for detecting SNP site marker, kit for detecting SNP site marker and application of SNP site marker and KASP primer

Abstract

The invention relates to a molecular biological technology, and discloses an SNP (Single Nucleotide Polymorphism) site marker in a tobacco genome, a KASP primer for detecting the SNP site marker, a kit for detecting the SNP site marker and application of the KASP primer and the kit. The invention discloses application of a reagent for detecting an SNP (Single Nucleotide Polymorphism) site marker in at least one of construction of a tobacco DNA (Deoxyribonucleic Acid) fingerprint spectrum, identification of tobacco bacterial wilt resistant germplasm resources and tobacco bacterial wilt resistant breeding, and the SNP site marker is at least one of ZXSNP1, ZXSNP2, ZXSNP3, ZXSNP4, ZXSNP5, ZXSNP6, ZXSNP7, ZXSNP8, ZXSNP9, ZXSNP10, ZXSNP11, ZXSNP12, ZXSNP13, ZXSNP14, ZXSNP15, ZXSNP16, ZXSNP17, ZXSNP18, ZXSNP19, ZXSNP20, ZXSNP21, ZXSNP22 The invention provides a set of credible KASP primers for SNP locus marking and detection, which are convenient for later use, for breeding development and research of tobacco bacterial wilt resistance, and provides a new resource and marker support for tobacco bacterial wilt resistance breeding work.

Description

Technical Field

[0001] The invention relates to molecular biology technology, in particular to SNP site markers in tobacco genomes, KASP primers for detection thereof, a detection kit and applications thereof. Background Art

[0002] Bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum, which is prevalent in tropical and subtropical regions. Ralstonia solanacearum has a wide host range and can infect plants from more than 50 families, including tobacco, potato, tomato, etc. Ralstonia solanacearum has a high degree of variability and adaptability, and there are obvious variations and differentiations in strains in different regions or between different hosts; the currently recognized typing methods mainly include physiological subspecies, biochemical types and phylotypes; Ralstonia solanacearum strains are mainly evolutionary type I, physiological subspecies 1 and 3, including 53 sequence variants; among them, the main one that infects tobacco is physiological subspecies 1, including 7 sequence variants.

[0003] At present, the occurrence of tobacco bacterial wilt is mainly suppressed by cultivation control, chemical control and biological control. Due to the limited effect of the above control methods and their environmental unfriendliness, breeding disease-resistant varieties is still a cost-effective method to control tobacco bacterial wilt. In the process of breeding resistant varieties, the excavation and utilization of excellent resistant germplasm resources is an important part. TI448A, which has been identified as an excellent flue-cured tobacco resource resistant to bacterial wilt, has become the main source of resistance to tobacco varieties resistant to bacterial wilt. Disease-resistant varieties bred accordingly include Oxford, DB101, DB102, DB27, Coker139, NC73, NC95, SC72, ​​Coker319, G28, etc. Among them, the bacterial wilt resistance of the main domestic tobacco germplasms K326, Yanyan 97 and DB101 also comes from TI448A. Therefore, the type of resistance to bacterial wilt appears to be very single, and the resistance line is easily broken by bacterial wilt. To this end, a large number of screening work has been carried out on tobacco bacterial wilt resistance germplasm resources, and local excellent disease-resistant germplasm resources such as Daye Mihe, GDSY-1, and Qiongzhong Wuzhishan have been identified. However, they are still in the stage of analyzing the resistance mechanism and have not yet been applied to tobacco bacterial wilt resistance breeding. Compared with the rich and diverse tobacco germplasm resources, tobacco germplasm resources resistant to bacterial wilt are still single. It is crucial to explore new excellent bacterial wilt resistance germplasm resources, especially the resistance sources of Chinese local varieties, and broaden the genetic background to promote the development of tobacco bacterial wilt breeding in China.

[0004] Molecular markers are closely linked to target trait genes and are a powerful weapon in the crop breeding process. With the advancement of biotechnology and sequencing methods, the third-generation molecular marker technology based on SNP (Single Nucleotide Polymorphism) has entered the high-throughput era, and DNA fingerprinting technology has emerged. Compared with traditional molecular markers, DNA fingerprints based on SNP molecular markers have obvious advantages in quantity, distribution range and stability, and are also widely used in the breeding of major crops such as rice, corn and wheat. In contrast, the construction of DNA fingerprints for tobacco germplasm resources resistant to bacterial wilt has not yet been effectively carried out. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem that the SNP molecular markers in the prior art have not been applied in the identification and certification of tobacco bacterial wilt resistant germplasm resources, and to provide SNP site markers in the tobacco genome and KASP primers for detection, a detection kit and application thereof.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides the use of a reagent for detecting SNP site markers in a tobacco genome in at least one of constructing a tobacco DNA fingerprint, identifying tobacco bacterial wilt-resistant germplasm resources, and tobacco bacterial wilt-resistant breeding, wherein the SNP site marker is at least one of ZXSNP1, ZXSNP2, ZXSNP3, ZXSNP4, ZXSNP5, ZXSNP6, ZXSNP7, ZXSNP8, ZXSNP9, ZXSNP10, ZXSNP11, ZXSNP12, ZXSNP13, ZXSNP14, ZXSNP15, ZXSNP16, ZXSNP17, ZXSNP18, ZXSNP19, ZXSNP20, ZXSNP21, ZXSNP22, ZXSNP23, and ZXSNP24;

[0007] The ZXSNP1 is located at position 63139808 on the chromosome Nt01 of tobacco, and its deoxynucleotide is C / A;

[0008] The ZXSNP2 is located at position 71000539 on the chromosome Nt02 of tobacco, and its deoxynucleotide is C / T;

[0009] The ZXSNP3 is located at position 92801818 on the chromosome Nt03 of tobacco, and its deoxynucleotide is A / G;

[0010] The ZXSNP4 is located at position 39871894 on the chromosome Nt04 of tobacco, and its deoxynucleotide is T / G;

[0011] The ZXSNP5 ​​is located at position 39276397 on the chromosome Nt05 of tobacco, and its deoxynucleotide is C / T;

[0012] The ZXSNP6 is located at position 46576280 on the chromosome Nt06 of tobacco, and its deoxynucleotide is G / T;

[0013] The ZXSNP7 is located at position 61124577 on the chromosome Nt07 of tobacco, and its deoxynucleotide is G / T;

[0014] The ZXSNP8 is located at position 16102461 on chromosome Nt08 of tobacco, and its deoxynucleotide is C / T;

[0015] The ZXSNP9 is located at position 45685160 on the chromosome Nt09 of tobacco, and its deoxynucleotide is C / G;

[0016] The ZXSNP10 is located at position 73932650 on the chromosome Nt10 of tobacco, and its deoxynucleotide is G / A;

[0017] The ZXSNP11 is located at position 36328237 on the chromosome Nt11 of tobacco, and its deoxynucleotide is C / T;

[0018] The ZXSNP12 is located at position 91531103 on the chromosome Nt12 of tobacco, and its deoxynucleotide is T / C;

[0019] The ZXSNP13 is located at position 23031905 on the chromosome Nt13 of tobacco, and its deoxynucleotide is A / C;

[0020] The ZXSNP14 is located at position 99385339 on the chromosome Nt14 of tobacco, and its deoxynucleotide is T / A;

[0021] The ZXSNP15 is located at position 88463594 on the chromosome Nt15 of tobacco, and its deoxynucleotide is G / A;

[0022] The ZXSNP16 is located at position 87894084 on the chromosome Nt16 of tobacco, and its deoxynucleotide is G / A;

[0023] The ZXSNP17 is located at position 184825986 on the chromosome Nt17 of tobacco, and its deoxynucleotide is C / G;

[0024] The ZXSNP18 is located at position 40740686 on the chromosome Nt18 of tobacco, and its deoxynucleotide is C / T;

[0025] The ZXSNP19 is located at position 52576511 on chromosome Nt19 of tobacco, and its deoxynucleotide is C / T;

[0026] The ZXSNP20 is located at position 103878491 on the chromosome Nt20 of tobacco, and its deoxynucleotide is C / T;

[0027] The ZXSNP21 is located at position 21153782 on the chromosome Nt21 of tobacco, and its deoxynucleotide is G / A;

[0028] The ZXSNP22 is located at position 101331353 on the chromosome Nt22 of tobacco, and its deoxynucleotide is G / A;

[0029] The ZXSNP23 is located at position 35126380 on the chromosome Nt23 of tobacco, and its deoxynucleotide is C / G;

[0030] The ZXSNP24 is located at position 56065989 on the chromosome Nt24 of tobacco, and its deoxynucleotide is T / C.

[0031] Preferably, the type of tobacco is selected from at least one of flue-cured tobacco, sun-cured tobacco, burley tobacco, cigar tobacco and oriental tobacco.

[0032] Preferably, the identification of tobacco germplasm resources resistant to bacterial wilt is the identification of cigar ZX145.

[0033] The second aspect of the present invention provides a set of KASP primers, which are KASP primers for detecting the above-mentioned SNP site markers;

[0034] The KASP primers corresponding to each SNP site marker to be tested include two forward primers and one reverse primer; the two forward primers are respectively recorded as forward primer 1 and forward primer 2; the 5' end of the forward primer 1 is connected to a fluorescent label sequence, and the 5' end of the forward primer 2 is connected to another fluorescent label sequence;

[0035] The nucleotide sequence of the forward primer 1 for detecting ZXSNP1 is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3;

[0036] The nucleotide sequence of the forward primer 1 for detecting ZXSNP2 is shown in SEQ ID No. 4, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 5, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6;

[0037] The nucleotide sequence of the forward primer 1 for detecting ZXSNP3 is shown in SEQ ID No.7, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.9;

[0038] The nucleotide sequence of the forward primer 1 for detecting ZXSNP4 is shown in SEQ ID No. 10, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 12;

[0039] The nucleotide sequence of the forward primer 1 for detecting ZXSNP5 ​​is shown in SEQ ID No. 13, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 14, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 15;

[0040] The nucleotide sequence of the forward primer 1 for detecting ZXSNP6 is shown in SEQ ID No. 16, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 18;

[0041] The nucleotide sequence of the forward primer 1 for detecting ZXSNP7 is shown in SEQ ID No. 19, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 20, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 21;

[0042] The nucleotide sequence of the forward primer 1 for detecting ZXSNP8 is shown in SEQ ID No. 22, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 23, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 24;

[0043] The nucleotide sequence of the forward primer 1 for detecting ZXSNP9 is shown in SEQ ID No. 25, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 26, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 27;

[0044] The nucleotide sequence of the forward primer 1 for detecting ZXSNP10 is shown in SEQ ID No. 28, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 30;

[0045] The nucleotide sequence of the forward primer 1 for detecting ZXSNP11 is shown in SEQ ID No. 31, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 32, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 33;

[0046] The nucleotide sequence of the forward primer 1 for detecting ZXSNP12 is shown in SEQ ID No. 34, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 35, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 36;

[0047] The nucleotide sequence of the forward primer 1 for detecting ZXSNP13 is shown in SEQ ID No. 37, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 38, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 39;

[0048] The nucleotide sequence of the forward primer 1 for detecting ZXSNP14 is shown in SEQ ID No. 40, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 41, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 42;

[0049] The nucleotide sequence of the forward primer 1 for detecting ZXSNP15 is shown in SEQ ID No. 43, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 44, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 45;

[0050] The nucleotide sequence of the forward primer 1 for detecting ZXSNP16 is shown in SEQ ID No. 46, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 47, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 48;

[0051] The nucleotide sequence of the forward primer 1 for detecting ZXSNP17 is shown in SEQ ID No. 49, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 50, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 51;

[0052] The nucleotide sequence of the forward primer 1 for detecting ZXSNP18 is shown in SEQ ID No. 52, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 53, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 54;

[0053] The nucleotide sequence of the forward primer 1 for detecting ZXSNP19 is shown in SEQ ID No.55, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.56, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.57;

[0054] The nucleotide sequence of the forward primer 1 for detecting ZXSNP20 is shown in SEQ ID No.58, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.59, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.60;

[0055] The nucleotide sequence of the forward primer 1 for detecting ZXSNP21 is shown in SEQ ID No.61, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.62, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.63;

[0056] The nucleotide sequence of the forward primer 1 for detecting ZXSNP22 is shown in SEQ ID No.64, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.65, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.66;

[0057] The nucleotide sequence of forward primer 1 for detecting ZXSNP23 is shown in SEQ ID No.67, the nucleotide sequence of forward primer 2 is shown in SEQ ID No.68, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.69;

[0058] The nucleotide sequence of the forward primer 1 for detecting ZXSNP24 is shown in SEQ ID No.70, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.71, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.72.

[0059] Preferably, the 5' end of the forward primer 1 is connected to a FAM fluorescent label sequence; and the 5' end of the forward primer 2 is connected to a HEX fluorescent label sequence.

[0060] Preferably, the FAM fluorescent tag sequence is shown as SEQ ID No.73, and the HEX fluorescent tag sequence is shown as SEQ ID No.74.

[0061] The third aspect of the present invention provides a kit, which contains the above-mentioned KASP primers.

[0062] The fourth aspect of the present invention provides the use of the above-mentioned KASP primers or the above-mentioned kit in at least one of constructing a tobacco DNA fingerprint, identifying tobacco bacterial wilt-resistant germplasm resources, and breeding tobacco bacterial wilt-resistant varieties.

[0063] A fifth aspect of the present invention provides a method for identifying tobacco germplasm resources resistant to bacterial wilt, the method comprising the following steps:

[0064] S1. Extract genomic DNA of all tobacco samples from the tested tobacco germplasm resources respectively;

[0065] S2. The tobacco sample DNA obtained in step S1 is subjected to PCR amplification using the corresponding primers in the above-mentioned KASP primers to specifically verify at least one of the above-mentioned SNP site markers.

[0066] Preferably, the type of the tobacco germplasm resources to be tested is at least one selected from flue-cured tobacco, sun-cured tobacco, burley tobacco, cigar tobacco and oriental tobacco.

[0067] Preferably, the identification of tobacco germplasm resources resistant to bacterial wilt is the identification of cigar ZX145.

[0068] Preferably, the specific verification process includes: detecting the FAM and HEX fluorescence values ​​of the PCR amplification product of the test tobacco sample DNA marked at the SNP site, and calculating the genotyping result of the test tobacco sample DNA marked at the SNP site.

[0069] Through the above technical scheme, the present invention provides a method of using at least one of the 24 specific SNP sites in the tobacco genome as a marker to efficiently identify tobacco germplasm with a high level of resistance to bacterial wilt, providing a set of reliable and easy-to-use SNP site markers and detection KASP primers for tobacco breeding development and research on bacterial wilt resistance, which can be used for the development of tobacco SNP detection kits, construction of tobacco DNA fingerprint maps, identification of tobacco bacterial wilt resistance germplasm resources, etc., providing new resources and marker support for tobacco bacterial wilt resistance breeding, and is particularly suitable for identifying cigar ZX145 with good bacterial wilt resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a graph of the resistance level of ZX145 in two different production areas of bacterial wilt disease nurseries in this application;

[0071] Figure 2 The chromosome distribution and quantity statistics of the ZX145-specific SNP loci in Example 1;

[0072] Figure 3 This is the typing of the ZX145-specific SNP fingerprint in 71 germplasm resources in Example 1. DETAILED DESCRIPTION

[0073] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0074] A first aspect of the present invention provides an application of a reagent for detecting SNP site markers in a tobacco genome in at least one of constructing a tobacco DNA fingerprint, identifying tobacco bacterial wilt-resistant germplasm resources, and tobacco bacterial wilt-resistant breeding, wherein the SNP site marker is at least one of ZXSNP1, ZXSNP2, ZXSNP3, ZXSNP4, ZXSNP5, ZXSNP6, ZXSNP7, ZXSNP8, ZXSNP9, ZXSNP10, ZXSNP11, ZXSNP12, ZXSNP13, ZXSNP14, ZXSNP15, ZXSNP16, ZXSNP17, ZXSNP18, ZXSNP19, ZXSNP20, ZXSNP21, ZXSNP22, ZXSNP23, and ZXSNP24;

[0075] The ZXSNP1 is located at position 63139808 on the chromosome Nt01 of tobacco, and its deoxynucleotide is C / A;

[0076] The ZXSNP2 is located at position 71000539 on the chromosome Nt02 of tobacco, and its deoxynucleotide is C / T;

[0077] The ZXSNP3 is located at position 92801818 on the chromosome Nt03 of tobacco, and its deoxynucleotide is A / G;

[0078] The ZXSNP4 is located at position 39871894 on the chromosome Nt04 of tobacco, and its deoxynucleotide is T / G;

[0079] The ZXSNP5 ​​is located at position 39276397 on the chromosome Nt05 of tobacco, and its deoxynucleotide is C / T;

[0080] The ZXSNP6 is located at position 46576280 on the chromosome Nt06 of tobacco, and its deoxynucleotide is G / T;

[0081] The ZXSNP7 is located at position 61124577 on the chromosome Nt07 of tobacco, and its deoxynucleotide is G / T;

[0082] The ZXSNP8 is located at position 16102461 on chromosome Nt08 of tobacco, and its deoxynucleotide is C / T;

[0083] The ZXSNP9 is located at position 45685160 on the chromosome Nt09 of tobacco, and its deoxynucleotide is C / G;

[0084] The ZXSNP10 is located at position 73932650 on the chromosome Nt10 of tobacco, and its deoxynucleotide is G / A;

[0085] The ZXSNP11 is located at position 36328237 on the chromosome Nt11 of tobacco, and its deoxynucleotide is C / T;

[0086] The ZXSNP12 is located at position 91531103 on the chromosome Nt12 of tobacco, and its deoxynucleotide is T / C;

[0087] The ZXSNP13 is located at position 23031905 on the chromosome Nt13 of tobacco, and its deoxynucleotide is A / C;

[0088] The ZXSNP14 is located at position 99385339 on the chromosome Nt14 of tobacco, and its deoxynucleotide is T / A;

[0089] The ZXSNP15 is located at position 88463594 on the chromosome Nt15 of tobacco, and its deoxynucleotide is G / A;

[0090] The ZXSNP16 is located at position 87894084 on the chromosome Nt16 of tobacco, and its deoxynucleotide is G / A;

[0091] The ZXSNP17 is located at position 184825986 on the chromosome Nt17 of tobacco, and its deoxynucleotide is C / G;

[0092] The ZXSNP18 is located at position 40740686 on the chromosome Nt18 of tobacco, and its deoxynucleotide is C / T;

[0093] The ZXSNP19 is located at position 52576511 on chromosome Nt19 of tobacco, and its deoxynucleotide is C / T;

[0094] The ZXSNP20 is located at position 103878491 on the chromosome Nt20 of tobacco, and its deoxynucleotide is C / T;

[0095] The ZXSNP21 is located at position 21153782 on the chromosome Nt21 of tobacco, and its deoxynucleotide is G / A;

[0096] The ZXSNP22 is located at position 101331353 on the chromosome Nt22 of tobacco, and its deoxynucleotide is G / A;

[0097] The ZXSNP23 is located at position 35126380 on the chromosome Nt23 of tobacco, and its deoxynucleotide is C / G;

[0098] The ZXSNP24 is located at position 56065989 on the chromosome Nt24 of tobacco, and its deoxynucleotide is T / C.

[0099] In the present invention, the physical position of the SNP site is determined based on the comparison of the tobacco K326 genome sequence (NCBI accession number: PRJNA376174). Specifically, the samtools and bcftools software are used to detect the population SNP of the sample, and the criteria for filtering the SNP site are as follows: take the SNP site with a sequencing of not less than 10X, and exclude the low-depth site; select the markers of the SNP site genotype covering all individuals; exclude the site with a MAF (Minor Allele Frequency) value lower than 0.05; filter out the site with a Het (Heterozygosity Rate) value greater than 0.6, and obtain all the SNP variation information of multiple tobacco germplasm resources with different resistance to bacterial wilt. ANNOVAR is used to perform functional annotation on the detected SNP variation sites, and according to the gene position of the SNP, it is divided into intergenic regions (intergenic), intronic regions (intronic) and exonic regions (exonic), etc., and the SNP site information between multiple germplasm resources is compared, and the specific SNP sites of tobacco germplasm with higher resistance to bacterial wilt are screened out.

[0100] According to the present invention, preferably, the type of tobacco is selected from at least one of flue-cured tobacco, sun-cured tobacco, burley tobacco, cigar tobacco and oriental tobacco. Further preferably, the identification of tobacco germplasm resources resistant to bacterial wilt is the identification of cigar tobacco ZX145, that is, by detecting the SNP locus markers provided by the present invention, cigar tobacco ZX145 with good bacterial wilt resistance is identified from the test tobacco germplasm resources.

[0101] The second aspect of the present invention provides a set of KASP primers, which are KASP primers for detecting the SNP site markers described in the first aspect above. The complete set of KASP primers can be used to detect all or part of the 24 SNP site markers, so as to specifically identify and distinguish tobacco germplasm resistance to bacterial wilt through PCR amplification of KASP primers.

[0102] Wherein, the KASP primers corresponding to each SNP site marker to be tested respectively include two forward primers and one reverse primer; wherein the two forward primers are respectively recorded as forward primer 1 and forward primer 2; the 5' end of the forward primer 1 is connected to a fluorescent label sequence, and the 5' end of the forward primer 2 is connected to another fluorescent label sequence;

[0103] The nucleotide sequence of the forward primer 1 for detecting ZXSNP1 is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3;

[0104] The nucleotide sequence of the forward primer 1 for detecting ZXSNP2 is shown in SEQ ID No. 4, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 5, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6;

[0105] The nucleotide sequence of the forward primer 1 for detecting ZXSNP3 is shown in SEQ ID No.7, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.9;

[0106] The nucleotide sequence of the forward primer 1 for detecting ZXSNP4 is shown in SEQ ID No. 10, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 12;

[0107] The nucleotide sequence of the forward primer 1 for detecting ZXSNP5 ​​is shown in SEQ ID No. 13, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 14, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 15;

[0108] The nucleotide sequence of the forward primer 1 for detecting ZXSNP6 is shown in SEQ ID No. 16, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 18;

[0109] The nucleotide sequence of the forward primer 1 for detecting ZXSNP7 is shown in SEQ ID No. 19, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 20, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 21;

[0110] The nucleotide sequence of the forward primer 1 for detecting ZXSNP8 is shown in SEQ ID No. 22, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 23, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 24;

[0111] The nucleotide sequence of the forward primer 1 for detecting ZXSNP9 is shown in SEQ ID No. 25, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 26, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 27;

[0112] The nucleotide sequence of the forward primer 1 for detecting ZXSNP10 is shown in SEQ ID No. 28, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 30;

[0113] The nucleotide sequence of the forward primer 1 for detecting ZXSNP11 is shown in SEQ ID No. 31, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 32, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 33;

[0114] The nucleotide sequence of the forward primer 1 for detecting ZXSNP12 is shown in SEQ ID No. 34, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 35, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 36;

[0115] The nucleotide sequence of the forward primer 1 for detecting ZXSNP13 is shown in SEQ ID No. 37, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 38, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 39;

[0116] The nucleotide sequence of the forward primer 1 for detecting ZXSNP14 is shown in SEQ ID No. 40, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 41, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 42;

[0117] The nucleotide sequence of the forward primer 1 for detecting ZXSNP15 is shown in SEQ ID No. 43, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 44, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 45;

[0118] The nucleotide sequence of the forward primer 1 for detecting ZXSNP16 is shown in SEQ ID No. 46, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 47, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 48;

[0119] The nucleotide sequence of the forward primer 1 for detecting ZXSNP17 is shown in SEQ ID No. 49, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 50, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 51;

[0120] The nucleotide sequence of the forward primer 1 for detecting ZXSNP18 is shown in SEQ ID No. 52, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 53, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 54;

[0121] The nucleotide sequence of the forward primer 1 for detecting ZXSNP19 is shown in SEQ ID No.55, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.56, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.57;

[0122] The nucleotide sequence of the forward primer 1 for detecting ZXSNP20 is shown in SEQ ID No.58, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.59, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.60;

[0123] The nucleotide sequence of the forward primer 1 for detecting ZXSNP21 is shown in SEQ ID No.61, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.62, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.63;

[0124] The nucleotide sequence of the forward primer 1 for detecting ZXSNP22 is shown in SEQ ID No.64, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.65, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.66;

[0125] The nucleotide sequence of forward primer 1 for detecting ZXSNP23 is shown in SEQ ID No.67, the nucleotide sequence of forward primer 2 is shown in SEQ ID No.68, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.69;

[0126] The nucleotide sequence of the forward primer 1 for detecting ZXSNP24 is shown in SEQ ID No.70, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.71, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.72.

[0127] According to the present invention, preferably, the 5' end of the forward primer 1 is connected to a FAM fluorescent tag sequence; the 5' end of the forward primer 2 is connected to a HEX fluorescent tag sequence. Further preferably, the FAM fluorescent tag sequence is shown in SEQ ID No.73, and the HEX fluorescent tag sequence is shown in SEQ ID No.74.

[0128] The third aspect of the present invention provides a kit, which contains the KASP primers provided in the second aspect.

[0129] In the present invention, the kit may also contain other reagents required for the PCR reaction system, such as Mastermix, MgCl2, ddH2O, etc.

[0130] Illustratively, the process of detecting SNP site markers using KASP primers includes: extracting genomic DNA of the tobacco to be tested, performing PCR amplification using the genomic DNA as a template using the KASP primers described in the above technical solution to obtain genotype data, and then constructing a tobacco DNA fingerprint map based on the genotype data, or analyzing and outputting genotype results to identify tobacco bacterial wilt-resistant germplasm resources and tobacco bacterial wilt-resistant breeding.

[0131] In the present invention, the PCR amplification procedure includes: 94°C pre-denaturation for 14 minutes; the first amplification reaction, 94°C denaturation for 20 seconds, 61-55°C extension for 60 seconds, 10 cycles; the second amplification reaction, 94°C denaturation for 20 seconds, 55°C extension for 60 seconds, 26 cycles. The PCR amplification system includes: 1.5 μL DNA template, 2.5 μL 2×KASP Master mix, 0.07 μL KASP Primermix, 0.93 μL water, and the total reaction system is 5 μL.

[0132] The fourth aspect of the present invention provides the use of the above-mentioned KASP primers or the above-mentioned kit in at least one of constructing a tobacco DNA fingerprint, identifying tobacco bacterial wilt-resistant germplasm resources, and breeding tobacco bacterial wilt-resistant varieties.

[0133] A fifth aspect of the present invention provides a method for identifying tobacco germplasm resources resistant to bacterial wilt, the method comprising the following steps:

[0134] S1. Extract genomic DNA of all tobacco samples from the tested tobacco germplasm resources respectively;

[0135] S2. The tobacco sample DNA obtained in step S1 is subjected to PCR amplification using the corresponding primers in the above-mentioned KASP primers, and specific verification is performed on at least one of the above-mentioned SNP site markers; preferably, specific verification is performed on 24 SNP site markers.

[0136] According to the present invention, preferably, the type of the test tobacco germplasm resources is selected from at least one of flue-cured tobacco, sun-cured tobacco, burley tobacco, cigar tobacco and oriental tobacco. In a specific embodiment of the present invention, the test tobacco is specifically selected from the 19 tobaccos shown in Table 1.

[0137] Further preferably, the identification of tobacco germplasm resources resistant to bacterial wilt is identification of Cigar ZX145, that is, Cigar ZX145 with good bacterial wilt resistance is identified from the tested tobacco germplasm resources by the method provided by the present invention.

[0138] According to the present invention, preferably, the specific verification process includes: detecting the FAM and HEX fluorescence values ​​of the PCR amplification product of the test tobacco sample DNA marked at the SNP site, and calculating the genotyping result of the test tobacco sample DNA marked at the SNP site.

[0139] Exemplarily, when a certain tobacco germplasm resource (e.g., cigar ZX145) is known to have resistance to bacterial wilt, the method for identifying tobacco germplasm resources resistant to bacterial wilt specifically includes: extracting genomic DNA from the test tobacco germplasm resource samples respectively; preparing a PCR reaction system for the test tobacco sample DNA in sequence according to the 24 SNP sites described above, and performing PCR amplification; using a KASP fluorescence detector to detect the FAM and HEX fluorescence values ​​of the PCR amplification product of the test tobacco sample DNA at each SNP site, and calculating the genotyping result of the test tobacco sample at the SNP site (i.e., detecting the specific nucleotide at the SNP site).

[0140] When the bacterial wilt resistance of a tobacco germplasm resource is unknown, the process of identifying whether it is a tobacco germplasm resource resistant to bacterial wilt may include: extracting the genomic DNA of the tobacco to be tested, detecting the genomic DNA using the SNP markers to obtain the genotype data of the tobacco to be tested; comparing the genotype data of the tobacco to be tested with the genotype data of tobacco with known low or no bacterial wilt resistance, and when there are more than three difference sites, the tobacco to be tested is a new tobacco germplasm resource resistant to bacterial wilt.

[0141] The present invention will be described in detail below through examples.

[0142] In the following examples, the test materials: 19 GBS (Genotyping-by-Sequencing) resequencing germplasm resources including cigar ZX145 (introduced from Brazil, high-quality multi-leaf wrapper, good resistance to bacterial wilt in the field) and other 18 tobacco germplasm resources; and 71 tobacco germplasm resources used for KASP primer verification are shown in Table 1.

[0143] All experimental materials were provided by the National Tobacco Germplasm Resources Medium-term Bank (Qingdao); unless otherwise specified, all reagents used were commercially available.

[0144] Table 1

[0145]

[0146]

[0147] The natural disease nursery resistance evaluation process of cigar tobacco ZX145: The materials used for the evaluation of bacterial wilt resistance include ZX145, susceptible control Honghua Dajinyuan (flue-cured tobacco, susceptible control), resistant control K326 (flue-cured tobacco, medium-resistant control), Yanyan 97 (flue-cured tobacco, resistant control), and DB101 (flue-cured tobacco, resistant control); the test sites for identification of bacterial wilt resistance were set up in the bacterial wilt disease nursery in Sanming, Fujian, the bacterial wilt disease nursery in Fuquan, Guizhou, and the bacterial wilt disease nursery in Xuancheng, Anhui. The soil fertility of the disease nursery was medium and uniform, the ground was flat, and the drainage and irrigation were convenient; the experiment was arranged in random blocks, with a total of 3 replicates, 20 plants planted in each replicate, and the plant spacing was 1.10m×0.40m; all test materials were uniformly raised and transplanted; the amount of fertilizer applied was calculated as pure nitrogen, 8.5kg per 667m2, and the N:P2O5:K2O ratio was 1:0.8:2.8; the field management measures referred to the local high-quality tobacco leaf production technology plan. Since the onset of the disease, the severity of the disease of the test materials was investigated every 10 days and the disease index was calculated according to the national standard GB / T 23222-2008. Excel (2021) and GraphPadPrism 8.0 were used for data processing, difference analysis and image drawing.

[0148] Disease grade is classified according to the following standards.

[0149] Level 0: Tobacco seedlings have no disease symptoms;

[0150] Level 1: 1 to 2 leaves are half wilted, or the chlorotic stripes on the stem occupy less than one-third of the plant height;

[0151] Level 2: 2 to 3 leaves wilt, or the stem has chlorotic streaks between one-third and one-half of the plant height;

[0152] Level 3: 1 to 2 healthy leaves, or chlorotic streaks on the stem at one-half to two-thirds of the plant height;

[0153] Level 4: The tobacco plant has no healthy leaves and is basically dead, or the stems have chlorotic streaks that exceed two-thirds of the plant height.

[0154] The disease index (DI) was calculated according to the following formula.

[0155]

[0156] According to the disease index of each tested variety, the disease resistance of the variety is divided into highly resistant (0), resistant (0.1<DI≤20), moderately resistant (20.1<DI≤40), moderately susceptible (40.1<DI≤60), susceptible (60.1<DI≤80), and highly susceptible (90<DI≤100).

[0157] The results are as follows Figure 1 As shown in the figure, in the natural disease nursery of Sanming, Fujian, the disease index of Cigarette ZX145 was 2.56, 7.02, 19.14, and 32.72, respectively, 10, 20, 30, and 40 days after the onset of bacterial wilt, which were all lower than those of the medium-resistant bacterial wilt materials K326, Yanyan 97, and DB101, and the bacterial wilt resistance reached the disease resistance level; in the natural disease nursery of Fuquan, Guizhou, the disease index of Cigarette ZX145 was 1.70, 11.10, 19.40, and 20.60, respectively, 10, 20, 30, and 40 days after the onset of bacterial wilt, which were higher than those of the medium-resistant bacterial wilt material K326 and the resistant bacterial wilt materials Yanyan 97 and DB101, and the bacterial wilt resistance reached the disease resistance level. In summary, Cigarette ZX145 showed good resistance in the three different production areas and had the value of breeding for bacterial wilt resistance.

[0158] Example 1

[0159] 1. SNP variation site analysis

[0160] The reference genome used for GBS resequencing data of 19 tobacco germplasm resources was the common tobacco K326 reference genome (NCBI accession number: PRJNA376174). The samtools and bcftools software were used to detect population SNPs in the samples. The criteria for filtering SNP sites were as follows: select SNP sites with sequencing no less than 10X and exclude low-depth sites; select markers whose SNP site genotypes cover all individuals; exclude sites with MAF (Minor Allele Frequency) values ​​less than 0.05; filter out sites with Het (Heterozygosity Rate) values ​​greater than 0.6, and obtain all SNP variation information of 19 tobacco germplasm resources. ANNOVAR was used to perform functional annotation on the detected SNP variation sites, and according to the gene location of the SNP, they were divided into intergenic regions, intronic regions, and exonic regions. The SNP site information between the 19 tobacco germplasm resources was compared, and the specific SNP sites of ZX145 were screened out.

[0161] GBS resequencing data of 19 tobacco germplasm resources obtained a total of 948.61Gbp of raw data, and after quality control, 945.17Gbp of clean data were obtained, with a sample GC range of 38.94-39.67%. The alignment rate of each sample with the K326 reference genome was between 94% and 95%, indicating that the sequencing data had high accuracy and reliability. According to the above-mentioned SNP variant site filtering criteria, 1,477,275 high-quality SNP sites were obtained from 19 tobacco germplasm resources, and the results are shown in Table 2. Among them, there are 292,848 SNP sites located inside the gene, 121058 SNP sites located in the coding region, 51047 sites leading to non-synonymous variation, 620 sites leading to premature termination variation (stop gain), and 129 sites leading to loss of stop codon variation (stop loss). According to the substitution types, there were 1,023,093 transitions (base transitions of G:A and C:T) and 454,182 transversions (base transitions of A:C, A:T, G:C and G:T), with a transition-to-transversion ratio of 2.252.

[0162] Table 2 Statistics of SNP annotation results

[0163]

[0164]

[0165] 2. Construction of ZX145-specific SNP fingerprint

[0166] By extracting and counting the specific SNPs of 19 germplasm resources, 221 ZX145-specific SNP loci were obtained. Figure 2 As shown in the figure, 221 specific SNP sites are distributed on 24 chromosomes, ranging from 2 in Nt01, Nt07 and Nt15 to 26 in Nt10 and Nt20, with an average of 9.2. There are 144 conversion-type SNP sites and 77 transversion-type SNP sites, with a conversion-transversion ratio of 1.87. Among the 221 specific SNP sites, there are 20 SNP sites located in genes, of which 5 are located in exon regions, 19 are located in the 1kb region upstream / downstream of genes, and the remaining 181 are located in intergenic regions. 1-2 ZX145-specific SNP sites were screened for each chromosome, and the SNP sites and 500bp upstream and downstream were extracted from the K326 reference genome for the development of KASP molecular markers.

[0167] For the specific SNP sites screened out, Tbtools software was used to extract the SNP sites and their 500bp upstream and downstream sequences from the K326 genome, and Primer 5.0 (https: / / www.premierbiosoft.com / ) was used to develop corresponding KASP markers for the specific SNP sites screened out; the primer design standards were: primer length 18-30bp, amplified fragment less than 250bp (including primers), GC content in the range of 20%-70%, TM value in the range of 58℃-64℃, the temperature difference between the three primers was controlled within 2℃, and the 3' end of the specific primer fell on the variant site. The KASP primers corresponding to each specific SNP site include two forward primers and one reverse primer. The 5' end of the forward primer 1 sequence is connected to the FAM fluorescent label sequence (as shown in SEQID No.73), and the 5' end of the forward primer 2 sequence is connected to the HEX fluorescent label sequence (as shown in SEQ ID No.74).

[0168] 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID No. 73);

[0169] 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID No. 74).

[0170] The designed KASP molecular markers were used to perform PCR amplification in 71 tobacco germplasm materials including flue-cured tobacco, cigar tobacco, wild tobacco, yellow flower tobacco, and burley tobacco as described in Table 1. The fluorescence signal was read using a TECAN infinite M1000 microplate reader, and then the online software snpdecoder (http: / / www.snpway.com / snpdecoder / ) was used to analyze the fluorescence signal to obtain a clear and intuitive typing diagram and output the genotype results. The analysis results were compared with the actual conditions of the varieties to determine the reliability of the fingerprint map.

[0171] Among them, the specific process of PCR amplification is:

[0172] (1) Preparation of DNA template: Genomic DNA was extracted from 71 samples of tobacco germplasm materials;

[0173] (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification was performed using the KASP primers designed above. The PCR reaction system is shown in Table 3. The PCR amplification procedure included: pre-denaturation at 94°C for 14 minutes; first step amplification reaction, denaturation at 94°C for 20 seconds, extension at 61-55°C for 60 seconds, and 10 cycles; second step amplification reaction, denaturation at 94°C for 20 seconds, extension at 55°C for 60 seconds, and 26 cycles.

[0174] Table 3 PCR reaction system

[0175] Components volume DNA template 1.5μL 2×KASP Master mix 2.5μL KASP Assay mix 0.07μL water 0.93μL Total volume 5μL

[0176] Among the 30 pairs of synthetically designed primers, 28 pairs could successfully complete genotyping, and the success rate of primer design was 93.3%. Based on the consideration of experimental cost optimization and chromosome coverage balance, one locus was selected for each chromosome, and the final 24 specific loci constituted the specific SNP fingerprint of ZX145, as shown in Table 4, and the corresponding KASP primers are shown in Table 5. The detection results of 71 germplasm resources (including ZX145) are shown in Figure 3 The results showed that the primer pairs of the 24 specific SNP loci could detect all samples completely and successfully distinguish ZX145 from the other 70 germplasm resources, all of which were homozygous genotypes. The 24 specific SNP loci and their KASP primers can provide an efficient tool for the identification and differentiation of ZX145 and other tobacco germplasm resources.

[0177] Table 4 ZX145 specific SNP fingerprint locus information

[0178] Serial number chromosome Physical Location SNP variation (other / ZX145) ZXSNP1 Nt01 63139808 C / A ZXSNP2 Nt02 71000539 C / T ZXSNP3 Nt03 92801818 A / G ZXSNP4 Nt04 39871894 T / G ZXSNP5 Nt05 39276397 C / T ZXSNP6 Nt06 46576280 G / T ZXSNP7 Nt07 61124577 G / T ZXSNP8 Nt08 16102461 C / T ZXSNP9 Nt09 45685160 C / G ZXSNP10 Nt10 73932650 G / A ZXSNP11 Nt11 36328237 C / T ZXSNP12 Nt12 91531103 T / C ZXSNP13 Nt13 23031905 A / C ZXSNP14 Nt14 99385339 T / A ZXSNP15 Nt15 88463594 G / A ZXSNP16 Nt16 87894084 G / A ZXSNP17 Nt17 184825986 C / G ZXSNP18 Nt18 40740686 C / T ZXSNP19 Nt19 52576511 C / T ZXSNP20 Nt20 103878491 C / T ZXSNP21 Nt21 21153782 G / A ZXSNP22 Nt22 101331353 G / A ZXSNP23 Nt23 35126380 C / G ZXSNP24 Nt24 56065989 T / C

[0179] Table 5 KASP primers for ZX145 specific SNP site markers

[0180]

[0181]

[0182]

[0183] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. Use of a reagent for detecting SNP site markers in tobacco genome in at least one of constructing tobacco DNA fingerprint, identifying tobacco bacterial wilt-resistant germplasm resources, and tobacco bacterial wilt-resistant breeding, wherein: The SNP site marker is at least one of ZXSNP1, ZXSNP2, ZXSNP3, ZXSNP4, ZXSNP5, ZXSNP6, ZXSNP7, ZXSNP8, ZXSNP9, ZXSNP10, ZXSNP11, ZXSNP12, ZXSNP13, ZXSNP14, ZXSNP15, ZXSNP16, ZXSNP17, ZXSNP18, ZXSNP19, ZXSNP20, ZXSNP21, ZXSNP22, ZXSNP23, and ZXSNP24; The ZXSNP1 is located at position 63139808 on the chromosome Nt01 of tobacco, and its deoxynucleotide is C / A; The ZXSNP2 is located at position 71000539 on chromosome Nt02 of tobacco, and its deoxynucleotide is C / T; The ZXSNP3 is located at position 92801818 on the chromosome Nt03 of tobacco, and its deoxynucleotide is A / G; The ZXSNP4 is located at position 39871894 on the chromosome Nt04 of tobacco, and its deoxynucleotide is T / G; The ZXSNP5 ​​is located at position 39276397 on the chromosome Nt05 of tobacco, and its deoxynucleotide is C / T; The ZXSNP6 is located at position 46576280 on the chromosome Nt06 of tobacco, and its deoxynucleotide is G / T; The ZXSNP7 is located at position 61124577 on the chromosome Nt07 of tobacco, and its deoxynucleotide is G / T; The ZXSNP8 is located at position 16102461 on chromosome Nt08 of tobacco, and its deoxynucleotide is C / T; The ZXSNP9 is located at position 45685160 on the chromosome Nt09 of tobacco, and its deoxynucleotide is C / G; The ZXSNP10 is located at position 73932650 on the chromosome Nt10 of tobacco, and its deoxynucleotide is G / A; The ZXSNP11 is located at position 36328237 on the chromosome Nt11 of tobacco, and its deoxynucleotide is C / T; The ZXSNP12 is located at position 91531103 on chromosome Nt12 of tobacco, and its deoxynucleotide is T / C; The ZXSNP13 is located at position 23031905 on the chromosome Nt13 of tobacco, and its deoxynucleotide is A / C; The ZXSNP14 is located at position 99385339 on the chromosome Nt14 of tobacco, and its deoxynucleotide is T / A; The ZXSNP15 is located at position 88463594 on the chromosome Nt15 of tobacco, and its deoxynucleotide is G / A; The ZXSNP16 is located at position 87894084 on the chromosome Nt16 of tobacco, and its deoxynucleotide is G / A; The ZXSNP17 is located at position 184825986 on the chromosome Nt17 of tobacco, and its deoxynucleotide is C / G; The ZXSNP18 is located at position 40740686 on the chromosome Nt18 of tobacco, and its deoxynucleotide is C / T; The ZXSNP19 is located at position 52576511 on chromosome Nt19 of tobacco, and its deoxynucleotide is C / T; The ZXSNP20 is located at position 103878491 on the chromosome Nt20 of tobacco, and its deoxynucleotide is C / T; The ZXSNP21 is located at position 21153782 on the chromosome Nt21 of tobacco, and its deoxynucleotide is G / A; The ZXSNP22 is located at position 101331353 on the chromosome Nt22 of tobacco, and its deoxynucleotide is G / A; The ZXSNP23 is located at position 35126380 on the chromosome Nt23 of tobacco, and its deoxynucleotide is C / G; The ZXSNP24 is located at position 56065989 on the chromosome Nt24 of tobacco, and its deoxynucleotide is T / C.

2. The use according to claim 1, characterized in that: The type of tobacco is selected from at least one of flue-cured tobacco, sun-cured tobacco, burley tobacco, cigar tobacco and oriental tobacco; Preferably, the identification of tobacco germplasm resources resistant to bacterial wilt is the identification of cigar ZX145.

3. A set of KASP primers, characterized in that: The set of KASP primers is a KASP primer used to detect the SNP site markers described in claim 1 or 2; The KASP primers corresponding to each SNP site marker to be tested include two forward primers and one reverse primer; the two forward primers are respectively recorded as forward primer 1 and forward primer 2; the 5' end of the forward primer 1 is connected to a fluorescent label sequence, and the 5' end of the forward primer 2 is connected to another fluorescent label sequence; The nucleotide sequence of the forward primer 1 used to detect ZXSNP1 is shown in SEQ ID No. 1, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 3; The nucleotide sequence of the forward primer 1 for detecting ZXSNP2 is shown in SEQ ID No. 4, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 5, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 6; The nucleotide sequence of the forward primer 1 for detecting ZXSNP3 is shown in SEQ ID No.7, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.9; The nucleotide sequence of the forward primer 1 for detecting ZXSNP4 is shown in SEQ ID No. 10, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 12; The nucleotide sequence of the forward primer 1 for detecting ZXSNP5 ​​is shown in SEQ ID No. 13, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 14, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 15; The nucleotide sequence of the forward primer 1 for detecting ZXSNP6 is shown in SEQ ID No. 16, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 18; The nucleotide sequence of the forward primer 1 for detecting ZXSNP7 is shown in SEQ ID No. 19, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 20, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 21; The nucleotide sequence of the forward primer 1 for detecting ZXSNP8 is shown in SEQ ID No. 22, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 23, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 24; The nucleotide sequence of the forward primer 1 for detecting ZXSNP9 is shown in SEQ ID No. 25, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 26, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 27; The nucleotide sequence of the forward primer 1 for detecting ZXSNP10 is shown in SEQ ID No. 28, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 30; The nucleotide sequence of the forward primer 1 for detecting ZXSNP11 is shown in SEQ ID No. 31, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 32, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 33; The nucleotide sequence of the forward primer 1 for detecting ZXSNP12 is shown in SEQ ID No. 34, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 35, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 36; The nucleotide sequence of the forward primer 1 for detecting ZXSNP13 is shown in SEQ ID No. 37, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 38, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 39; The nucleotide sequence of the forward primer 1 for detecting ZXSNP14 is shown in SEQ ID No. 40, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 41, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 42; The nucleotide sequence of the forward primer 1 for detecting ZXSNP15 is shown in SEQ ID No. 43, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 44, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 45; The nucleotide sequence of the forward primer 1 for detecting ZXSNP16 is shown in SEQ ID No. 46, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 47, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 48; The nucleotide sequence of the forward primer 1 for detecting ZXSNP17 is shown in SEQ ID No. 49, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 50, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 51; The nucleotide sequence of the forward primer 1 for detecting ZXSNP18 is shown in SEQ ID No. 52, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No. 53, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 54; The nucleotide sequence of the forward primer 1 for detecting ZXSNP19 is shown in SEQ ID No.55, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.56, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.57; The nucleotide sequence of the forward primer 1 for detecting ZXSNP20 is shown in SEQ ID No.58, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.59, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.60; The nucleotide sequence of the forward primer 1 for detecting ZXSNP21 is shown in SEQ ID No.61, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.62, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.63; The nucleotide sequence of the forward primer 1 for detecting ZXSNP22 is shown in SEQ ID No.64, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.65, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.66; The nucleotide sequence of forward primer 1 for detecting ZXSNP23 is shown in SEQ ID No.67, the nucleotide sequence of forward primer 2 is shown in SEQ ID No.68, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.69; The nucleotide sequence of the forward primer 1 for detecting ZXSNP24 is shown in SEQ ID No.70, the nucleotide sequence of the forward primer 2 is shown in SEQ ID No.71, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.

72.

4. The KASP primer according to claim 3, characterized in that The 5' end of the forward primer 1 is connected to a FAM fluorescent label sequence; the 5' end of the forward primer 2 is connected to a HEX fluorescent label sequence.

5. The KASP primer according to claim 4, characterized in that The FAM fluorescent tag sequence is shown in SEQ ID No.73, and the HEX fluorescent tag sequence is shown in SEQ ID No.

74.

6. A kit, characterized in that: The kit contains the KASP primer according to any one of claims 3 to 5.

7. Use of the KASP primers according to any one of claims 3 to 5 or the kit according to claim 6 in at least one of constructing tobacco DNA fingerprints, identifying tobacco bacterial wilt-resistant germplasm resources, and breeding tobacco bacterial wilt-resistant varieties.

8. A method for identifying tobacco germplasm resources resistant to bacterial wilt, characterized in that: The method comprises the following steps: S1. Extract genomic DNA of all tobacco samples from the tested tobacco germplasm resources respectively; S2. The tobacco sample DNA obtained in step S1 is subjected to PCR amplification using the corresponding primers in the KASP primers described in any one of claims 3 to 5 to specifically verify at least one of the SNP site markers in claim 1.

9. The method according to claim 8, characterized in that The type of the tobacco germplasm resources to be tested is selected from at least one of flue-cured tobacco, sun-cured tobacco, burley tobacco, cigar tobacco and oriental tobacco; Preferably, the identification of tobacco germplasm resources resistant to bacterial wilt is the identification of cigar ZX145.

10. The method according to claim 8, characterized in that The specific verification process includes: detecting the FAM and HEX fluorescence values ​​of the PCR amplification product of the test tobacco sample DNA marked at the SNP site, and calculating and obtaining the genotyping result of the test tobacco sample DNA marked at the SNP site.

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