SNP (Single Nucleotide Polymorphism) site combination for tobacco germplasm resource identification and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an SNP site combination for tobacco germplasm resource identification and application of the SNP site combination. According to the invention, 396 tobacco germplasm resource materials are collected, and a set of 96 SNP locus combination with high quality and high polymorphism is developed based on re-sequencing data; the 96 SNP site combination is used for variety identification, genetic diversity analysis, purity identification and fingerprint spectrum analysis of tobacco genetic materials, the coverage degree is higher, the accuracy is good, germplasm resource distinguishing is more accurate, the genotype detection quality is more stable, efficient and accurate distinguishing of seed resources in tobacco breeding is facilitated, and rapid breeding is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to SNP locus combinations for the identification of tobacco germplasm resources and their applications. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) is an important model plant and also a significant economic crop. Developing high-quality tobacco leaves is essential for the sustainable development of tobacco production. Breeding superior tobacco varieties is widely recognized as the most effective and fundamental way to improve tobacco quality. Accelerating the cultivation of excellent new tobacco varieties and promoting the sustainable development of the tobacco industry are of great importance.

[0003] Traditional tobacco breeding relies primarily on experience-based phenotypic selection, which, while achieving good breeding results, is labor-intensive, time-consuming, inefficient, and overly dependent on the environment. In recent years, breeding methods have evolved from traditional morphological and cytological markers to high-throughput molecular markers, whole-genome sequencing, and phenomics technologies, significantly improving the accuracy and scope of genetic diversity analysis. However, morphological markers are susceptible to environmental interference, cytological markers are complex to operate, and biochemical markers have limited polymorphism. In contrast, molecular marker technology, through DNA-level polymorphism detection, offers advantages such as high resolution, high reproducibility, and whole-genome coverage, and has become a core tool for genetic diversity research, occupying a mainstream position in germplasm resource evaluation, population genetic structure analysis, and molecular breeding.

[0004] SNP markers are currently widely used molecular markers. However, current SNP molecular markers based on tobacco germplasm breeding have problems such as insufficient material representativeness and poor ability to distinguish tobacco types. Therefore, developing a set of core SNP molecules with higher coverage and stronger distinguishing ability for tobacco germplasm resource identification can improve the throughput and efficiency of tobacco germplasm resource identification and variety approval, and lay a good foundation for the comprehensive identification and evaluation of tobacco germplasm resources in my country, systematic tobacco resource classification, and fingerprinting construction. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide SNP locus combinations and their applications for identification of tobacco germplasm resources, genetic diversity analysis, purity identification and / or fingerprint analysis.

[0006] This invention provides a combination of SNP loci for identification of tobacco germplasm resources, genetic diversity analysis, purity identification and / or fingerprint analysis, which includes 96 SNP loci as shown in Table 1.

[0007] This invention provides primer combinations that target the SNP site combinations described herein.

[0008] Furthermore, this invention utilizes the KASP detection system to design primer combinations based on the KASP detection system for tobacco germplasm resource identification, genetic diversity analysis, purity identification, and / or fingerprint analysis. The primer combinations include primer C with the nucleotide sequence shown in SEQ ID NO: (n-1), primer X with the nucleotide sequence shown in SEQ ID NO: n, and primer Y with the nucleotide sequence shown in SEQ ID NO: (n+1).

[0009] Where n is any integer between 2 and 287, with an interval of 3. Specifically, in a specific embodiment of the present invention, n is 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 1 52, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284 and / or 287.

[0010] Furthermore, primer X has a fluorescent group attached to its 5' end, and primer Y has a fluorescent group attached to its 5' end, with the fluorescent groups attached to the 5' ends of primer X and primer Y being different.

[0011] In a specific embodiment of the present invention, the fluorescent group is selected from FAM and HEX.

[0012] This invention provides reagents for identifying tobacco germplasm resources, analyzing genetic diversity, determining purity, and / or analyzing fingerprints, comprising the primer combinations described herein. Further, the reagents comprise the primer combinations described herein and a solvent; the solvent may be a specific buffer system, which is not limited thereto by this invention.

[0013] This invention provides a kit for identifying tobacco germplasm resources, analyzing genetic diversity, determining purity, and / or analyzing fingerprints, comprising:

[0014] At least one of nucleic acid extraction reagents and nucleic acid amplification reagents and the primer combination described in this invention; or

[0015] At least one of nucleic acid extraction reagents and nucleic acid amplification reagents, and the reagents described in this invention.

[0016] Furthermore, the nucleic acid extraction reagent includes lysis buffer, washing buffer and / or elution buffer; the amplification reagent includes DNA polymerase, dNTPs and / or amplification buffer, etc.

[0017] This invention provides at least one of the following applications (I) to IV) in tobacco germplasm resource identification, genetic diversity analysis, purity identification, and / or fingerprint analysis:

[0018] I) The SNP site combination described in this invention;

[0019] II) The primer combination described in this invention;

[0020] III) The reagents described in this invention;

[0021] IV) The reagent kit described in this invention.

[0022] This invention provides a method for identifying tobacco germplasm resources, analyzing genetic diversity, determining purity, and / or analyzing fingerprint patterns, comprising detecting tobacco samples using at least one of the following methods (A) to (D):

[0023] A) The SNP site combination described in this invention;

[0024] B) The primer combination described in this invention;

[0025] C) The reagents described in this invention;

[0026] D) The reagent kit described in this invention.

[0027] Furthermore, the method described in this invention includes the following steps:

[0028] After extracting DNA from the sample, it was tested using the kit described in this invention.

[0029] This invention collected 396 Chinese tobacco germplasm resources and, based on resequencing data, clearly defined screening thresholds: heterozygosity <0.3, deletion rate <0.1, MAF >0.2, and sequencing depth >5x. From the resequencing data of these 396 germplasm resources, 19,615 candidate loci were precisely screened. After primer design feasibility analysis, 4,895 high-quality loci were finally obtained. Further screening yielded the 96 core SNP locus combinations shown in this invention. The advantages of these 96 SNP locus combinations for tobacco germplasm resource identification are as follows:

[0030] (i) Genetic diversity is more comprehensive and adaptability is wider.

[0031] 1. Enhanced representativeness of germplasm materials: This invention is based on resequencing of 396 Chinese tobacco germplasm resources (covering major types such as flue-cured tobacco, sun-cured tobacco, burley tobacco, and aromatic tobacco). The large sample size ensures the population representativeness of SNP loci. Compared with SNP loci sets developed based on local germplasm resources, this invention has broader coverage and higher adaptability.

[0032] 2. More balanced and in-depth chromosome coverage: The 96 core SNP loci of this invention are evenly distributed across the 24 chromosomes of tobacco, with 4 high-quality loci precisely selected from each chromosome, covering key functional regions of the entire genome. Compared with some existing SNP locus sets, the coverage density is significantly improved, resulting in a higher detection rate for key genetic variation regions that are easily missed.

[0033] 3. Superior ability to distinguish between different types of tobacco: This invention was verified through fingerprint analysis of 16 different types of tobacco germplasm, and all materials achieved 100% accurate differentiation with no cases of confusion; the cluster analysis results of 38 germplasm samples were completely consistent with the actual pedigree and ecological type. Compared with some SNP locus sets in existing technologies, it is more accurate and effective in distinguishing some closely related varieties, and the differentiation accuracy is significantly improved.

[0034] (ii) Genotyping quality is more stable and detection reliability is higher.

[0035] 1. Significant advantages in the quantitative analysis of key indicators: The detection rate of all 96 SNP sites in this invention is higher than 99%, and the homozygotes and heterozygotes are clustered compactly and clearly classified, with no ambiguous sites.

[0036] 2. Stronger anti-interference ability: This invention effectively eliminates unstable sites that are susceptible to environmental influences by using strict site screening criteria (heterozygosity < 0.3, deletion rate < 0.1); compared with the existing SNP site set, the detection of some low polymorphism and low stability sites is more accurate, and the genotyping is clearer.

[0037] (III) Significant advantages in detection efficiency and throughput

[0038] This invention's SNP site detection is compatible with Douglas Scientific's Array Tape high-throughput platform, offering high automation, low reagent consumption (only 0.8 μL per reaction), and a cost reduction of over 30%, meeting the needs of large-scale germplasm resource screening. The comparative document uses a 384-well PCR instrument with a reaction volume of 6 μL, exhibiting lower throughput and cost control than this invention, and does not mention optimization for high-throughput detection platforms.

[0039] This invention collected 396 tobacco germplasm resources and developed a set of 96 high-quality, highly polymorphic SNP loci combinations based on resequencing data. These 96 SNP loci combinations are used for variety identification, genetic diversity analysis, and purity identification of tobacco genetic materials. The fingerprint analysis has higher coverage, better accuracy, more precise germplasm resource differentiation, and more stable genotype detection quality, which is beneficial for the efficient and accurate differentiation of seed resources in tobacco breeding and for achieving rapid breeding. Attached Figure Description

[0040] Figure 1 Genotyping diagram of the KASP marker Chr1-13619922 in 48 tobacco materials;

[0041] Figure 2 Cluster analysis diagram of 38 tobacco materials is shown;

[0042] Figure 3 Fingerprint spectra of 16 tobacco materials are shown. Detailed Implementation

[0043] This invention provides SNP locus combinations for tobacco germplasm resource identification and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0044] In the detection of the 96 SNP site combinations, this invention features a more complete primer design and validation process, resulting in higher specificity.

[0045] 1. More comprehensive primer design tools and analysis dimensions: This invention uses BatchPrimer3 online software to systematically analyze four key indicators for 300bp sequences upstream and downstream of SNP sites: GC content (optimized range 40%~60%), copy number (single copy preferred), primer dimer, hairpin structure, etc., to ensure primer amplification specificity.

[0046] 2. More rigorous primer verification: This invention optimizes 4895 designed sites, excluding sites with low amplification efficiency and poor specificity, and finally selects 4 sites with the best quality for each chromosome to ensure that all 96 pairs of primers can amplify stably.

[0047] The sequencing and variant detection method based on the combined detection of the aforementioned 96 SNP sites is more advanced and produces better data quality.

[0048] 1. More accurate variant detection process: This invention adopts the process of "Reads alignment - single sample gVCF generation - joint-calling joint analysis", which can effectively integrate population-level variant information and reduce false positive variants in single sample detection; the comparison file only uses haplotype block analysis and data formatting, without involving population joint analysis, which has a higher risk of false positive variants.

[0049] 2. Better fit of the reference genome: This invention uses the tobacco K326 reference genome (currently the most widely used and most well-annotated tobacco reference genome) to ensure the accuracy of SNP site location.

[0050] 3. More stringent quality control of sequencing data: This invention uses resequencing data with a sequencing depth of 10X and a sequencing volume of 50Gb to ensure the accuracy and coverage of SNP sites; the comparative document does not specify the sequencing depth and data volume, and only screens 1,179,154 SNP sites from 150 germplasms, so the reliability and representativeness of the data basis are slightly inferior.

[0051] This invention provides a clear standard for the selection of superior traits in tobacco using SNP sites.

[0052] (a) Genetic diversity criteria: ensuring broad coverage of markers

[0053] Key metrics: The screening criteria were heterozygosity <0.3, site deletion rate <0.1, MAF >0.2, and sequencing depth >5x.

[0054] (ii) Variety-specific standards: ensuring the distinguishing accuracy of the markings.

[0055] Key indicator: There is at least one difference in core SNP loci among different varieties;

[0056] Experimental verification: The fingerprint spectrum analysis of 16 tobacco samples by this invention showed that all materials could be distinguished with 100% accuracy.

[0057] Other key technical features of this invention that play a crucial role in the detection of the aforementioned 96 SNP site combinations:

[0058] (I) Targeted optimization of PCR amplification system and conditions

[0059] More efficient amplification system: This invention uses Douglas Scientific's Array Tape system for automated PCR assembly, with a single reaction volume of only 0.8 μL (containing primers, Master Mix, and DNA template); and the amount of DNA template used is only 20~50 ng, making it more suitable for the detection of precious germplasm materials.

[0060] More precise amplification conditions: The PCR amplification conditions of the present invention are "94°C for 15 min; 94°C for 20 s, 65°C to 57°C (the annealing temperature decreases by 0.8°C per cycle) for 60 s, 10 cycles; 94°C for 20 s, 57°C for 60 s, 30 cycles". The annealing temperature gradient is more reasonable and the number of cycles is more optimized, effectively improving the primer specific amplification efficiency.

[0061] (2) Full-process optimization of DNA extraction and quality control

[0062] The present invention has established a standardized DNA extraction process suitable for high-throughput detection: using freeze-drying, high-throughput tissue grinding (1400 rpm × 3 min), CTAB warm bath (65°C, for 1 - 1.5 h), magnetic bead purification, and automated extraction by ME480 instrument to ensure DNA purity (260 / 280 = 1.8 - 2.2) and integrity (the main band on 1% agarose gel electrophoresis is clear without degradation).

[0063] (3) Customized adaptation of data analysis and genotyping software

[0064] The present invention uses the INTELLICS software supporting the Array Tape system for genotype typing. The software has optimized the algorithm for the fluorescence signal characteristics of KASP markers, can automatically identify homozygous and heterozygous clustering, and the genotyping accuracy rate is over 99.5%.

[0065] (4) Expansive design of marker application scenarios

[0066] The 96 SNP markers of the present invention are not only applicable to germplasm resource identification and fingerprint map construction, but can also be directly used for: ① rapid detection of variety purity; ② parental selection in genetic breeding (rapidly screening parental combinations based on genetic distance); ③ authenticity identification in variety right protection (can distinguish varieties with extremely close genetic relationships).

[0067] The present invention provides a set of SNP detection methods based on KASP markers. The designed primer pairs are applicable to multiple platforms such as high-throughput SNP genotyping platforms and high-throughput KASP detection platforms, and can be applied to large-scale population breeding and analysis.

[0068] The present invention provides a KASP marker detection method based on the Douglas Array Tape platform. The Douglas Array Tape platform has high automation, high throughput, high speed, less reagent consumption, and low detection cost.

[0069] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with the embodiments:

[0070] Example 1

[0071] 1. Collection of resequencing data from tobacco germplasm resources

[0072] To ensure the representativeness of tobacco materials, resequencing data of 396 Chinese tobacco germplasm resources (covering major types such as flue-cured tobacco, sun-cured tobacco, burley tobacco, and aromatic tobacco) were collected. The sequencing depth of each sample was 10X and the sequencing volume was 50Gb.

[0073] 2. Comparison and variant detection were performed on 396 resequencing data.

[0074] The analysis process is as follows:

[0075] (1) Align the reads to the corresponding tobacco reference genome (K326), sort the positions and mark duplicate reads.

[0076] (2) Detect the mutation sites for each sample and obtain the gVCF of each sample.

[0077] (3) Perform joint-calling to conduct joint analysis of gVCF for all samples to obtain the variation results of each individual in the population.

[0078] 3. Core site screening

[0079] a. Candidate site screening: SNP variant sites were extracted from the resequencing data. The screening criteria included heterozygosity <0.3, site deletion rate <0.1, minimum allele frequency of MAF >0.2, and sequencing depth >5x. A total of 19,615 SNP polymorphic sites were screened as candidate sites.

[0080] b. Site primer design and screening: 300bp sequences upstream and downstream of SNP sites were extracted and analyzed for GC content, copy number, primer dimer, etc., and 4895 SNP sites that could be used for marker development were screened.

[0081] 4. KASP primer design

[0082] KASP primers were designed for the 4895 high-quality loci selected using the online software BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). The KASP marker consisted of three primers: two allele-specific primers, X (Primer_X) and Y (Primer_Y), and one universal primer, C (Primer_C). The 5' ends of the allele-specific primers were connected to the LGC KASP reaction-specific fluorescent groups FAM and HEX, respectively. If only FAM fluorescence was detected in the sample, the genotype was homozygous allele X (Allele_X); if only HEX fluorescence was detected, the genotype was homozygous allele Y (Allele_Y); if both FAM and HEX fluorescence were detected, the genotype was heterozygous (carrying both alleles X and Y).

[0083] The GC content, copy number, and primer dimer of the primer sequences were evaluated. Four sites with the best quality and high-quality primer sequences were selected from each chromosome for primer synthesis. A total of 96 sets of primers were synthesized, as shown in Table 1.

[0084] Table 1. Allelic types and primer sequences of KASP markers

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] 5. Development of SNP molecular markers for tobacco

[0092] The 96 loci were designed and developed into a set of SNP molecular markers based on KASP (Kompetitive Allele Specific PCR) technology.

[0093] This technology involves the following steps:

[0094] (1) Sample DNA extraction and quality control:

[0095] ① Take 20~30mg of material and put it into a 96-well plate, then freeze dry it using a freeze dryer;

[0096] ② Use a bead separator to add two 4mm steel balls to the 96-hole plate of the vacuum-evacuated blade, and cover it with the matching silicone film.

[0097] ③ Place the 96-well plate in a high-throughput tissue grinder, adjust the speed to 1400 rpm and grind for 3 minutes. The grinding time can be increased until the leaf is crushed.

[0098] ④ Take out the 96-well plate and add 600 μL of CTAB extraction solution to each well with a pipette. After sealing with heat-sealing film, place it on a vortex shaker and shake well to mix.

[0099] ⑤ Place the sealed 96-well plate in a water bath at 65°C for 1 to 1.5 hours, taking it out several times during this period and placing it on a vortex shaker to mix it properly.

[0100] ⑥ After the warm bath, remove the sealed 96-well plate and place it in a refrigerated centrifuge. Set the speed to 4000 rpm and the temperature to 4°C, and centrifuge for 10 minutes.

[0101] ⑦ Remove the centrifuged 96-well plate and use a semi-automatic 96-well pipette to transfer 400 μL of the supernatant to 2 mL of purified 96-well plate;

[0102] ⑧ Add an equal volume of magnetic bead mixing solution to a 2 mL purification 96-well plate containing 400 μL, and place it in the ME480 extraction instrument;

[0103] ⑨ Place the washing buffer plate in positions 2-3 after extraction, and place the DNA-dissolving plate containing 150 μL of elution buffer in position 4.

[0104] ⑩ Run the ME-480 extraction program. The extraction process takes about 30 minutes. Cover the DNA plate with a membrane for storage. The extraction process is now complete.

[0105] The DNA samples were analyzed using a UV spectrophotometer, requiring a sample concentration greater than 10 ng / μL and a purity between 1.8 and 2.2 for the 260 / 280 ratio. The integrity of the DNA samples was assessed using 1% agarose gel electrophoresis (gel concentration: 1%; voltage: 120V; electrophoresis time: 25 min; loading volume: 1 μL), requiring a clearly defined main band and no significant degradation. Samples that passed quality control were used for library preparation.

[0106] (2) KASP tag verification:

[0107] KASP-tagged reactive sequencing was performed using the Douglas Scientific ArrayTape system. The ArrayTape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0108] PCR reaction system: The PCR amplification system was automatically assembled using NEXAR, and the PCR reaction system is shown in Table 2 below.

[0109] Table 2. PCR reaction system for KASP marker genotyping

[0110] PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: 94℃ for 15 min; 94℃ for 20 s, 65℃~57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 s, 10 cycles; 94℃ for 20 s, 57℃ for 60 s, 30 cycles.

[0111] Signal scanning and genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then genotyping and data analysis were performed using INTELLICS.

[0112] 6. Results Display

[0113] (1) Genotyping detection of 96 KASP markers:

[0114] Ninety-six KASP markers were validated using 48 diverse tobacco samples. In KASP marker genotyping, the samples were divided into three genotypes: homozygous genotype cluster X, cluster Y, and heterozygous genotype cluster XY (see...). Figure 1 The X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (marked in red in the upper left corner of the genotyping graph), the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (marked in blue in the lower right corner of the genotyping graph), and the heterozygous genotype cluster indicates that the sample contains heterozygous X and Y alleles at this KASP marker locus (marked in purple in the genotyping graph). Verification showed that the two homozygous and heterozygous clusters for each KASP marker were well-generated and compact, with single-copy loci and detection rates exceeding 99%. The genotyping quality of the 96 KASP markers fully meets the requirements for accurate detection of tobacco samples. The genotyping graph for the Chr1-13619922 marker is shown below. Figure 1 .

[0115] (2) Cluster analysis of tobacco materials

[0116] Genotyping of 38 tobacco germplasm resources was performed using 96 core SNP loci selected from the study. Genotypic data were extracted, and genetic distance matrices were calculated using Plink software. Cluster analysis was then performed to construct a phylogenetic tree, which can determine the kinship, evolutionary relationships, and compositional structure of different materials. Population analysis of the 38 tobacco materials using the 96 SNP molecular markers of this invention yielded results consistent with actual population grouping. This indicates that the selected SNP loci are highly representative. Figure 2 .

[0117] (3) Fingerprint analysis of tobacco materials

[0118] Genotyping was performed on 16 tobacco germplasm resources using 96 core SNP loci selected from screening. Genotyping was extracted and fingerprint analysis was performed. Based on the SNP genotyping data generated by KASP detection, DNA fingerprints were constructed for each germplasm resource material. Each material contains 96 core SNP loci, each locus consisting of two bases. Each column represents one SNP marker, and each row represents one material. Red represents homozygous XX (ref-ref), blue represents homozygous YY (alt-alt), and green represents heterozygous XY (ref-alt).

[0119] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of SNP loci for use in the identification of tobacco germplasm, the analysis of genetic diversity, the identification of purity and / or the analysis of a fingerprint, characterized in that, comprise 96 SNP loci as shown in Table 1.

2. A primer combination targeting the combination of SNP loci according to claim 1.

3. The primer combination according to claim 2, characterized in that, The primer combination comprises a primer C having a nucleotide sequence as shown in SEQ ID NO: (n-1), a primer X having a nucleotide sequence as shown in SEQ ID NO: n and a primer Y having a nucleotide sequence as shown in SEQ ID NO: (n+1). wherein n is all integers between 2 and 287 at intervals of 3.

4. The primer combination according to claim 3, characterized in that, The 5' end of the primer X is connected with a fluorescent group, the 5' end of the primer Y is connected with a fluorescent group, and the fluorescent groups connected to the 5' ends of the primer X and the primer Y are different.

5. Reagent, characterized in that, comprise the primer combination according to any one of claims 2 to 4.

6. A kit, characterized in that, comprise: at least one of a nucleic acid extraction reagent and a nucleic acid amplification reagent and the primer combination according to any one of claims 2 to 4; or at least one of a nucleic acid extraction reagent and a nucleic acid amplification reagent and the reagent according to claim 5. The nucleic acid extraction reagent comprises a lysis solution, a washing solution and / or an elution solution.

8. Use of at least one of the following I) to IV) in tobacco germplasm identification, genetic diversity analysis, purity identification and / or fingerprinting analysis:

7. The kit of claim 6, wherein I) the combination of SNP loci according to claim 1; II) the primer combination according to any one of claims 2 to 4; III) the reagent according to claim 5; IV) the kit according to claim 6 or 7. comprise detecting a tobacco sample using at least one of the following A) to D): A) the combination of SNP loci according to claim 1; 9. A method for the identification of tobacco germplasm resources, genetic diversity analysis, purity identification and / or fingerprinting analysis, characterized in that, B) the primer combination according to any one of claims 2 to 4; C) the reagent according to claim 5; D) the kit according to claim 6 or 7. comprise detecting after extracting DNA of a sample using the kit according to claim 6 or 7. ​ 10. The method of claim 9, wherein, ​

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