A SNP marker method for efficiently distinguishing multiple tobacco varieties

Abstract

The application discloses a SNP marker method for efficiently distinguishing a plurality of tobacco varieties, wherein the SNP marker is 8, each marker comprises 3 primer groups, and FAM and HEX fluorescent linker sequences are connected to the 5' ends of two specific primers respectively. The primer sequences of the SNP marker are shown as SEQ ID No. 1-24. The molecular marker, the primer group or the kit containing the primer group can be applied in the following fields: identifying tobacco variety resources, constructing a tobacco variety DNA fingerprint, molecular assisted breeding of tobacco varieties, authenticity identification of tobacco leaves, tobacco variety tracing and the like. The SNP molecular marker for efficiently identifying seven Yunnan tobacco varieties is provided, the marker is genotyped by using the KASP technology principle, is good in specificity, is low in cost, is simple to operate, and can realize automatic high-throughput detection by matching an automatic instrument.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an efficient SNP labeling method for distinguishing multiple tobacco varieties. Background Technology

[0002] my country is the world's largest producer of tobacco leaves, and tobacco plays a vital role in the national economy. Superior varieties are crucial for the development of flue-cured tobacco production, representing an intrinsic factor in obtaining high-quality tobacco leaves and a relatively economical and effective measure for increasing yield, quality, and income. my country has the world's largest collection of tobacco germplasm resources, and new tobacco varieties suitable for planting in approved regions are constantly emerging. There is a need for tobacco variety differentiation in germplasm resource identification, tobacco breeding, and flue-cured tobacco production. Tobacco variety differentiation mainly employs morphological identification, physiological and biochemical index analysis, and molecular marker technology. However, conducting uniqueness, uniformity, and stability (DUS) tests on tobacco varieties through large-scale field planting presents challenges such as long cycles, high difficulty, high labor intensity, and susceptibility to environmental influences. Physiological and biochemical index analysis generally requires specialized equipment and personnel, and the available indicators are limited, with poor polymorphism among varieties. SSR molecular marker technology, due to its high polymorphism, co-dominance, and stable inheritance advantages, has been applied to crop variety identification, but it suffers from drawbacks such as limited quantity, low throughput, and environmental unfriendliness, making it insufficient to meet the demands of today's large-scale testing. SNP molecular markers are third-generation molecules that determine genetic differences between individuals by detecting variations at SNP loci. They have the advantages of high stability and high-throughput detection and have been widely used in the identification of variety authenticity and purity testing of crops such as rice, wheat, corn, and soybeans. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide an efficient SNP marker method for distinguishing multiple tobacco varieties. It can distinguish seven representative tobacco varieties from Yunnan and can be combined with automated instruments to achieve high-throughput and low-cost detection. It is suitable for applications such as molecular-assisted breeding of tobacco varieties, variety identification of flue-cured tobacco, and authenticity identification of tobacco leaves.

[0004] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0005] A method for efficiently distinguishing multiple tobacco varieties using SNP markers, comprising eight SNP markers, the information of which is as follows:

[0006]

[0007] Preferably, in the above technical solution, each marker includes three primers, namely allele-specific primers X and Y and universal primer C, wherein the 5' ends of the two specific primers are respectively connected to FAM and HEX fluorescent adapter sequences.

[0008] Preferably, in the above technical solution, the primer sequence is as follows:

[0009] The primer sequences for Nt901740_K01 are shown in SEQ ID No. 1-3;

[0010] The primer sequences for Nt901749_K01 are shown in SEQ ID No. 4-6;

[0011] The primer sequences for Nt901754_K01 are shown in SEQ ID No. 7-9;

[0012] The primer sequences for Nt901758_K01 are shown in SEQ ID No. 10-12;

[0013] The primer sequences for Nt901766_K01 are shown in SEQ ID No. 13-15;

[0014] The primer sequences for Nt901767_K01 are shown in SEQ ID No. 16-18;

[0015] The primer sequences for Nt901775_K01 are shown in SEQ ID No. 19-21;

[0016] The primer sequence for Nt901776_K01 is shown in SEQ ID No. 22-24.

[0017] A kit for efficiently distinguishing multiple tobacco varieties, the kit comprising a primer set.

[0018] Preferably, the above technical solution further includes: reagents for DNA extraction, a reaction system for PCR amplification, and KASP reaction reagents for genotyping detection.

[0019] Any of the following applications of a molecular marker, primer set, or kit:

[0020] (1) Application in identifying tobacco varietal resources;

[0021] (2) Application in constructing DNA fingerprint profiles of tobacco varieties;

[0022] (3) Application in molecular-assisted breeding of tobacco varieties;

[0023] (4) Application in the identification of the authenticity of tobacco leaves;

[0024] (5) Application in tobacco variety tracing.

[0025] A method for identifying multiple tobacco varieties, the method comprising the following steps:

[0026] (1) Extract DNA from the tobacco sample to be tested;

[0027] (2) Use primer sets for PCR amplification, use a scanner to detect fluorescence of the amplification products, and determine the genotype of the sample based on the fluorescence detection results.

[0028] Preferably, in the above technical solution, if only the fluorescence signal corresponding to specific primer X is detected in the PCR product of the sample, the genotype of the detection site is allele X; if only the fluorescence signal corresponding to specific primer Y is detected, the genotype of the detection site is allele Y; if both fluorescence signals are detected simultaneously, the genotype of the detection site is heterozygous.

[0029] The above-described technical solution of the present invention has the following beneficial effects:

[0030] This invention provides a set of efficient SNP molecular markers for identifying seven Yunnan tobacco varieties. These markers utilize the KASP technology principle for genotyping, exhibiting high specificity, low cost, and ease of operation. When combined with automated instruments, they can achieve automated high-throughput detection. Attached Figure Description

[0031] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0032] Figure 1 This is a flowchart of the SNP labeling method for efficiently distinguishing tobacco varieties according to the present invention.

[0033] Figure 2 This is a diagram showing the genotyping effect of the SNP marker genotype in this invention. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0035] Unless otherwise specified, all reagents used in this application are commercially available or obtained through commercial channels, or may be prepared by referring to existing chemical methods.

[0036] Example 1: SNP tag screening

[0037] Whole-genome resequencing was performed on seven major tobacco varieties cultivated in Yunnan in recent years using BGI's high-throughput sequencing platform. The steps are as follows:

[0038] (1) Extract tobacco genomic DNA using a nucleic acid extraction kit.

[0039] (2) Using the MGI standard method, construct a 300-500bp DNA fragment library.

[0040] (3) After the library passes the test, it is sequenced using the PE150 sequencing strategy and the sequencing depth is 10×, with 40Gb sequenced for each variety.

[0041] (4) After the sequencing data is processed, it is filtered, sequencing data contamination is detected, and sequencing data quality is assessed. After the resequencing data passes quality control, it is compared with the reference genome for variant site analysis and resequencing is calculated.

[0042] The quality indicators for sequencing sites were as follows: heterozygosity <0.05, deletion rate <0.05, sequencing depth >10×, and polymorphic SNP sites in at least two varieties were selected as candidate sites. Candidate SNP sites were screened according to the requirements for marker development, and finally, eight core SNP markers for tobacco varieties were developed, as shown in Table 1.

[0043] Table 1: Tag Information

[0044]

[0045] Example 2

[0046] A method for identifying seven Yunnan tobacco varieties using core SNP markers from eight SNP tobacco varieties.

[0047] (1) DNA extraction

[0048] Tobacco genomic DNA was extracted from cured tobacco leaves using a magnetic bead method. High-throughput DNA extraction was performed using an automated workstation.

[0049] A. Add 4mm steel balls to the sample well plate, cover with a silicone cap, and grind at 1400rpm for 2min;

[0050] B. After grinding, add 400 μL of lysis buffer into the well plate and incubate at 65°C for 1 hour;

[0051] C. After warm bath, centrifuge at 3600 rpm for 10 min, place in LGC automated magnetic bead extractor, and run the plant DNA extraction process program;

[0052] D. Confirm that there are no errors during the program's operation, complete the DNA extraction process, and seal and store the DNA for later use.

[0053] (2) Primer design and synthesis

[0054] Polymorphic sites were identified from resequencing data of seven representative varieties from Yunnan Province. Flanking sequences of SNP sites were extracted, and primers were designed using the online primer design website BatchPrimer3. Each set of primers was labeled with three primers, with two specific primers having FAM and HEX fluorescent adapter sequences attached to their 5' ends, respectively. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primers are shown in Table 2.

[0055] Table 2 Primer Sequences

[0056]

[0057] (3) PCR amplification (KASP reaction test)

[0058] KASP reaction assays were performed on the Douglas genotyping platform. 20 ng of DNA sample was added to each microplate, dried, and then KASP reaction mixture was added. The reaction system is shown in Table 3. PCR amplification was performed in a water bath thermal cycler. The Touchdown PCR reaction conditions were: 94℃ pre-denaturation for 15 minutes; first amplification reaction: 94℃ denaturation for 20 seconds, annealing and extension at 65℃–57℃ for 60 seconds, 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ per cycle; second amplification reaction: 94℃ denaturation for 20 seconds, annealing and extension at 57℃ for 60 seconds, 32 cycles. After the reaction, the KASP reaction products were scanned for fluorescence data, and the fluorescence scan results were automatically converted into images.

[0059] The Douglas genotyping platform and its accompanying reagents and consumables used in this invention were purchased from LGC, UK.

[0060] Table 3: Reaction system for KASP detection

[0061]

[0062] (4) Labeling and typing data

[0063] SNP markers based on the KASP reaction principle were designed, with each marker consisting of three primers: two specific primers with fluorescent adapter sequences from LGC's KASP reaction reagent attached to their 5' ends, and one universal primer. If only the fluorescence signal corresponding to primer PrimerX was detected in the PCR product of the sample, the genotype of the detected site was Allele X; if only the fluorescence signal corresponding to primer PrimerY was detected, the genotype of the detected site was Allele Y; if both fluorescence signals were detected simultaneously, the genotype of the detected site was heterozygous.

[0064] Genotyping of tobacco DNA was performed using SNP markers. Eight markers were selected that could completely distinguish seven tobacco varieties. The genotyping data are shown in Table 4.

[0065] Table 4: Genotypes of 7 tobacco varieties

[0066] High-quality molecular marker validation can effectively identify flue-cured tobacco varieties, providing a scientific basis and reference for variety identification, variety ownership confirmation, and genetic purity verification. Core SNP markers can be tandemly combined with more molecular markers for marker-assisted breeding and variety improvement in tobacco. Furthermore, phylogenetic tree analysis, principal component analysis, and genetic similarity analysis can be used to evaluate the 10-factor identification ability of the obtained SNP markers, and varietal genomics data, phenotypic information, marker cohorts, and other information can be stored in a database for easy resource sharing and sample backtracking.

[0067] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A KASP primer set for SNP markers used to efficiently distinguish tobacco varieties, characterized in that, Each marker includes three primers: allele-specific primers X and Y, and a universal primer C. The 5' ends of the two allele-specific primers are connected to FAM and HEX fluorescent adapter sequences, respectively. The specific nucleotide sequences of the primer set are shown in SEQ ID No. 1-24.

2. The KASP primer set for efficiently distinguishing seven tobacco varieties using SNP markers according to claim 1, characterized in that, The primer sequences are as follows: The primer sequences for Nt901740_K01 are shown in SEQ ID No. 1-3; The primer sequences for Nt901749_K01 are shown in SEQ ID No. 4-6; The primer sequences for Nt901754_K01 are shown in SEQ ID No. 7-9; The primer sequences for Nt901758_K01 are shown in SEQ ID No. 10-12; The primer sequences for Nt901766_K01 are shown in SEQ ID No. 13-15; The primer sequences for Nt901767_K01 are shown in SEQ ID No. 16-18; The primer sequences for Nt901775_K01 are shown in SEQ ID No. 19-21; The primer sequence for Nt901776_K01 is shown in SEQ ID No. 22-24.

3. A reagent kit for efficiently distinguishing seven or more tobacco varieties, characterized in that, The kit contains the primer set as described in any one of claims 1-2.

4. The reagent kit according to claim 3, characterized in that, It also includes: reagents for DNA extraction, reaction systems for PCR amplification, and KASP reaction reagents for genotyping.

5. Any of the following applications of the primer set according to any one of claims 1-2 or the kit according to any one of claims 3-4: (1) Application in the identification of seven tobacco varieties: Yun 87, Yun 97, Yun 100, Yun 105, Yun 116, Yun 121, and Honghua Dajinyuan; (2) Application in constructing DNA fingerprint profiles of seven tobacco varieties: Yun87, Yun97, Yun100, Yun105, Yun116, Yun121, and Honghua Dajinyuan; (3) Application in the identification of the authenticity of tobacco leaves of seven tobacco varieties: Yun 87, Yun 97, Yun 100, Yun 105, Yun 116, Yun 121, and Honghua Dajinyuan; (4) Application in the varietal tracing of seven tobacco varieties: Yun 87, Yun 97, Yun 100, Yun 105, Yun 116, Yun 121, and Honghua Dajinyuan; The tobacco varieties mentioned are seven types: Yun 87, Yun 97, Yun 100, Yun 105, Yun 116, Yun 121, and Honghua Dajinyuan.

6. A method for identifying seven tobacco varieties: Yun 87, Yun 97, Yun 100, Yun 105, Yun 116, Yun 121, and Honghua Dajinyuan, characterized in that... Includes the following steps: (1) Extract DNA from the tobacco sample to be tested; (2) PCR amplification is performed using the primer set as described in any one of claims 1-2, and the amplification products are detected by fluorescence using a scanner. The genotype of the sample is determined based on the fluorescence detection results.

7. The method for identifying multiple tobacco varieties according to claim 6, characterized in that, If only the fluorescence signal corresponding to specific primer X is detected in the PCR product of the sample, the genotype of the detection site is allele X; if only the fluorescence signal corresponding to specific primer Y is detected, the genotype of the detection site is allele Y; if both fluorescence signals are detected simultaneously, the genotype of the detection site is heterozygous.

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