KASP molecular marker for drought resistance character of tobacco and application of KASP molecular marker

Abstract

The invention relates to the technical field of biology, in particular to a KASP molecular marker for tobacco drought resistance characters and application of the KASP molecular marker. The molecular marker c22-1 and the molecular marker c22-4 which are closely linked with the tobacco drought resistance character are finally obtained by carrying out a first round of screening on the tobacco drought resistance character gene based on sequencing data and then carrying out a second round of large-scale screening on the tobacco drought resistance character gene by utilizing a KASP technology. The molecular marker is used for breeding the drought-resistant tobacco variety, the detection result stability is good, the repeatability is high, the detection result consistency is good, a more accurate scientific basis can be provided for subsequent gene function research and breeding work, and the breeding process of the drought-resistant tobacco variety is accelerated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the KASP molecular marker for drought resistance in tobacco and its application. Background Technology

[0002] Tobacco thrives in warm, sunny conditions and is highly dependent on water. Its growth and development are extremely susceptible to environmental influences, and drought is particularly detrimental to tobacco production, severely restricting yield and quality and causing enormous losses to tobacco production nationwide and globally. Enhancing tobacco's resilience and thus improving its yield stability has become a crucial research topic for the stable development of the tobacco industry. Therefore, exploring drought-resistant germplasm and genetic resources and cultivating new drought-resistant varieties (lines) is urgently needed.

[0003] KASP, as a mainstream high-throughput SNP technology, has achieved high-precision genotyping of biallelic genes (SNPs and InDels) due to its excellent stability and accuracy. Therefore, identifying functional SNP sites related to drought resistance in tobacco based on sequencing and RNA-seq data, and developing KASP molecular markers for tobacco drought resistance suitable for high-throughput molecular detection platforms, has significant application value for improving the breeding efficiency and level of drought-resistant tobacco varieties, and will greatly promote the progress of gene mapping and molecular-assisted breeding. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a KASP molecular marker for the drought resistance trait in tobacco and its application.

[0005] This invention provides SNP sites associated with drought resistance traits in tobacco, including C22-1 SNP sites and / or C22-4 SNP sites;

[0006] In this invention, the C22-1 SNP site is located at position 109518286 on the 22nd chromosome of tobacco, and the polymorphism is T / C;

[0007] The C22-4 SNP site is located at position 110249618 on chromosome 22 of the tobacco plant, and its polymorphism is A / G.

[0008] This invention screened four candidate regions related to the trait (the genomic locations of the four candidate regions are: chr22:109406636:110090379, chr22:110237277:110388698, chr22:110602420:110692956, and sca54:50771:688613) by analyzing BSA pooled sequencing and RNA-seq data from the parents. The regions were 1.56 MB in length and contained 33 transcripts, representing candidate segments of 33 genes. KASP technology was then used to further screen the candidate segments. After screening, c22-1 and c22-4 were identified as molecular markers closely linked to the drought resistance trait in tobacco.

[0009] This invention provides a primer set targeting the SNP sites described herein.

[0010] Furthermore, the primer set targeting the C22-1 SNP site includes: a competitive specific forward primer 1 with a nucleotide sequence as shown in SEQ ID NO:1, a competitive specific forward primer 2 with a nucleotide sequence as shown in SEQ ID NO:2, and a universal reverse primer with a nucleotide sequence as shown in SEQ ID NO:5;

[0011] The primer set targeting the C22-4 SNP site includes: a competitive specific forward primer 1 with the nucleotide sequence shown in SEQ ID NO:6, a competitive specific forward primer 2 with the nucleotide sequence shown in SEQ ID NO:7, and a universal reverse primer with the nucleotide sequence shown in SEQ ID NO:5.

[0012] Furthermore, the 5' end of the competitive specific forward primer 1 and / or the competitive specific forward primer 2 is fluorescently labeled;

[0013] The fluorescent labels of the competitive specific forward primer 1 and / or the competitive specific forward primer 2 are different.

[0014] Specifically, the fluorescent labels include: FAM, HEX, ROX, Cy5 and / or JOE.

[0015] In a specific embodiment of the present invention, the fluorescent label of the competitive specific forward primer 1 is FAM; and the fluorescent label of the competitive specific forward primer 2 is HEX.

[0016] The present invention provides a reagent containing at least one of a stabilizer, a solvent, a preservative, and the primer set described in the present invention.

[0017] This invention provides a kit for breeding drought-resistant tobacco varieties, which includes the reagents described in this invention and auxiliary detection reagents or devices.

[0018] Furthermore, the reagents used for auxiliary detection include PCR amplification reagents.

[0019] This invention provides the application of at least one of the following (I) to (IV) in the breeding of drought-resistant tobacco varieties:

[0020] I) The SNP sites described in this invention;

[0021] II) The primer set described in this invention;

[0022] III) The reagents described in this invention;

[0023] IV) The reagent kit described in this invention.

[0024] This invention provides a method for breeding drought-resistant tobacco varieties, characterized in that the tobacco is detected using the SNP locus described in claim 1 as a target, or by using at least one of the following methods (i) to (iii), and then the drought resistance is determined based on the detection results:

[0025] i) The primer set described in this invention;

[0026] ii) The reagents described in this invention;

[0027] iii) The reagent kit described in this invention.

[0028] Furthermore, the criteria for judging whether something is drought-resistant are as follows:

[0029] If the C22-1 SNP site is homozygous C, then it indicates drought resistance;

[0030] If the C22-1 SNP site is homozygous T, then it is drought-sensitive;

[0031] If the C22-4 SNP site is homozygous for G, then it indicates drought resistance;

[0032] If the C22-4 SNP site is homozygous A, then it is drought-sensitive.

[0033] This invention, based on sequencing data, first screens tobacco drought-resistance genes and then uses KASP technology for a second round of large-scale screening. Finally, it obtains two molecular markers closely linked to tobacco drought resistance traits: c22-1 and c22-4. These molecular markers are used for breeding drought-resistant tobacco varieties. The detection results show good stability, high repeatability, and good consistency, providing more accurate scientific evidence for subsequent gene function research and breeding work, and accelerating the breeding process of drought-resistant tobacco varieties. Attached Figure Description

[0034] Figure 1 The stability and repeatability of C22-1 and C22-4 markers are shown in Figure A; the stability and repeatability of C22-1 markers are shown in Figure B, where Allele 1 / Allele1 is TT, Allele 2 / Allele 2 is CC, and Allele 1 / Allele 2 is CT; the stability and repeatability of C22-4 markers are shown in Figure B, where Allele 1 / Allele1 is AA, Allele 2 / Allele 2 is GG, and Allele 1 / Allele 2 is GA.

[0035] Figure 2 Scatter plots showing allele typing for C22-1 and C22-4 markers; A represents the C22-1 locus, where Allele 1 / Allele1 is TT, Allele 2 / Allele 2 is CC, and Allele 1 / Allele 2 is CT; B represents the C22-4 locus, where Allele 1 / Allele1 is AA, Allele 2 / Allele 2 is GG, and Allele 1 / Allele 2 is GA.

[0036] Figure 3 Both C22-4 and C22-1 can be used to identify drought-resistant genotypes in the K326 and Nanjiang 3 populations; A represents the C22-1 locus, where Allele 1 / Allele1 is TT, Allele 2 / Allele 2 is CC, and Allele 1 / Allele 2 is CT; B represents the C22-4 locus, where Allele 1 / Allele1 is AA, Allele 2 / Allele 2 is GG, and Allele 1 / Allele 2 is GA. Detailed Implementation

[0037] This invention provides KASP molecular markers for drought resistance traits in tobacco 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 realize and apply the technology of this invention.

[0038] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0039] Example 1: A KASP molecular marker for drought resistance in tobacco and its application

[0040] I. Development of KASP molecular markers for drought resistance in tobacco

[0041] The molecular markers obtained in this invention are molecular markers that are closely linked to the drought resistance trait in tobacco, and can be used for molecular-assisted selection breeding to screen different materials and their offspring.

[0042] Based on BSA pooled sequencing and RNA-seq data, we located tobacco drought resistance genes, searched for single nucleotide polymorphism (SNP) sites in target genomic regions, and developed functional KASP molecular markers closely related to drought resistance traits. Specific steps and parameters are as follows:

[0043] For progeny variant sites filtered by parental markers (K326 and Nanjiang 3), the index value of each site in the wild-type pool and the ∆SNP-index value between pools were first calculated separately. To reduce the random fluctuations caused by single variant sites, sliding window and Loess fitting methods were used to denoise the ∆SNP-index values. To improve the accuracy of the analysis, the 0.995 quantile was used as the threshold for the sliding window results to obtain candidate regions. For Loess fitting, the 0.995 quantile was used as the threshold to obtain candidate regions. To avoid errors caused by uneven labeling, each candidate region was required to contain at least 10 variant sites above the threshold line. The sliding window-based denoising algorithm yielded four trait-related candidate regions, each 1.56 MB in length, containing 33 transcripts and 33 genes.

[0044] After the target region was located by BSA, candidate genes in the target region were initially screened using KASP technology. KASP primers were designed by pre-selecting sites with no other variant sites within 30 bp upstream and downstream, and where the sequences within 50 bp above and below the site did not have other highly similar sequences in the reference genome. The KASP design parameters were: (1) maximum annealing temperature of 63℃; (2) maximum primer length of 25 bp. A total of 14 pairs of KASP primers were designed.

[0045] It has been verified that, compared with other SNP sites, C22-1 and C22-4 can serve as molecular markers closely linked to the drought resistance trait in tobacco.

[0046] Specifically, the genomic locations and related information of the C22-1 and C22-4 molecular markers are as follows:

[0047] C22-1: chr22, position 109518286, drought-sensitive (T / T), drought-resistant (C / C), heterozygous between drought-sensitive and drought-resistant;

[0048] c22-4: chr22, position 110249618, A / A drought-sensitive, G / G drought-resistant, heterozygous between drought-sensitive and drought-resistant.

[0049] For the tobacco reference genome, please see the link: http: / / lifenglab.hzau.edu.cn / Nicomics / Download / index.php.

[0050] KASP primers were designed targeting the pre- and post-suppression sequences of the target SNPs C22-1 and C22-4, including two competitive specific forward primers and one universal reverse primer. Each forward primer has a corresponding SNP allele at its 3' end and a tag sequence for binding to different fluorescent markers added to its 5' end. Genotypes were determined based on the fluorescence signal carried by the PCR product; homozygous genotypes produced only one fluorescence signal, while heterozygous genotypes produced mixed fluorescence signals. Two corresponding KASP molecular markers were developed, named C22-1 and C22-4, respectively. The primer sequences for amplifying these KASP molecular markers are as follows:

[0051] C22-1-F1: 5'-cctaagactctagagcagccA-3', SEQ ID NO: 1;

[0052] C22-1-F2: 5'-cctaagactctagagcagccG-3', SEQ ID NO: 2;

[0053] C22-1-F1: 5'-GAAGGTGACCAAGTTCATGCTcctaagactctagagcagccA-3', SEQ ID NO: 3, the bold uppercase part of the 5' end is the fluorescent tag FAM, which is a green fluorescent signal;

[0054] C22-1-F2: 5'-GAAGGTCGGAGTCAACGGATTcctaagactctagagcagccG-3', SEQ ID NO:4, the sequence of the bold uppercase letters at the 5' end is the fluorescent tag HEX;

[0055] C22-1-R: 5'-cccactgctttggattttcgtG-3', SEQ ID NO: 5;

[0056] C22-4-F1: 5'-ctctcaagcatgcttctgcA-3', SEQ ID NO: 6;

[0057] C22-4-F2: 5'-ctctcaagcatgcttctgcG-3', SEQ ID NO: 7, the sequence of the bold uppercase letter part at the 5' end is the fluorescent tag HEX;

[0058] C22-4-F1: 5'-GAAGGTGACCAAGTTCATGCTctctcaagcatgcttctgcA-3', SEQ ID NO: 8, the sequence of the bold uppercase letters at the 5' end is the fluorescent tag FAM;

[0059] C22-4-F2: 5'-GAAGGTCGGAGTCAACGGATTctctcaagcatgcttctgcG-3', SEQ ID NO: 9, the sequence of the bold uppercase letter part at the 5' end is the fluorescent tag HEX;

[0060] C22-4-R: 5'-aggggtgaaatacaggtgcg-3', SEQ ID NO: 10;

[0061] II. Stability and repeatability of C22-1 and C22-4 markers

[0062] First, homozygous parents K326 and Nanjiang 3, exhibiting differences in the target trait, were selected for constructing the segregating population. At least three independent biological replicates were prepared for each parent to assess the stability and reproducibility of the C22-1 and C22-4 markers. The specific steps are as follows:

[0063] High-quality genomic DNA was extracted from young leaves of parental plants using a commercially available kit (such as the QIAGEN DNeasy Plant Mini Kit). The concentration and purity of the DNA were measured using a NanoDrop microspectrophotometer to ensure an A260 / A280 ratio between 1.8 and 2.0. All DNA samples were uniformly diluted to a working concentration of 20 ng / μL. The following 10 μL reaction mixture was prepared: 5 μL 2×KASP Master Mix (containing optimized Taq DNA polymerase, dNTPs, MgCl2, and a proprietary fluorescent dye), 1 μL primer premix (containing equimolar concentrations of C22-1 and C22-4 allele-specific primers F1 / F2 and universal primer R, as shown in SEQ ID NO: 3-5, 8-10), and 20 ng template DNA, to be made up with sterile water.

[0064] Run the following program on a real-time fluorescence PCR instrument (such as Bio-Rad CFX384, Applied Biosystems QuantStudio): Hot start: 94°C, 15 min (activation of hot-start enzyme); Touchdown amplification (10 cycles): 94°C, 20 s (denaturation); 65–57°C, 60 s (annealing / extension), decreasing by 0.6°C per cycle. This step improves the specificity of primer binding to the target sequence; Standard amplification (26–30 cycles): 94°C, 20 s; 55°C, 60 s. After the annealing / extension phase of the last cycle, the instrument automatically acquires the fluorescence signal. The ABI 7500 instrument software monitors the growth of both FAM and HEX / VIC fluorescence signals in real time. After the reaction, the software performs automatic cluster analysis based on the endpoint fluorescence signal.

[0065] The results showed that the two parents were clearly grouped into two distinct clusters, and the biological and technical repetitions of the same parent were tightly clustered within the same cluster, indicating that the markers had high repeatability and reliability. Figure 1 Tables 1 and 2 (X is...) Figure 1 The horizontal axis is Y, which is Figure 1 (Middle vertical axis).

[0066] Table 1. Repeatability verification of C22-1 marker

[0067]

[0068] Table 2. Repeatability verification of C22-4 marker

[0069]

[0070] III. Consistency Analysis of C22-1 and C22-4 Marker Detection Results

[0071] The accuracy and reliability of the C22-1 and C22-4 markers were verified in drought-resistant and drought-sensitive materials from the K326 and Nanjiang 3 F2 populations whose phenotypes had been identified. Results showed that the C22-1 and C22-4 markers accurately matched the phenotypes and genotypes of the materials. The C22-1 marker results showed that 7 out of 16 drought-resistant materials had a homozygous CC genotype, consistent with the drought-resistant parent Nanjiang 3, while the remaining 9 had a heterozygous CT genotype. Similarly, 4 out of 18 drought-sensitive materials had a homozygous TT genotype, consistent with the non-drought-resistant parent K326, while the remaining 14 had a heterozygous CT genotype. The C22-4 marker results showed that 7 out of 16 drought-resistant materials had a homozygous GG genotype, consistent with the drought-resistant parent Nanjiang 3, while the remaining 9 had a heterozygous GA genotype. Likewise, 4 out of 18 drought-sensitive materials had a homozygous AA genotype, consistent with the non-drought-resistant parent K326, while the remaining 14 had a heterozygous GA genotype. The results of the C22-1 and C22-4 markers showed a very high degree of consistency, meaning that the genotypes of the plants were consistent.

[0072] The results are as follows Figure 2 ( Figure 2 The results shown include, in addition to the plants used for verification, two homozygous parents (Tables 3 and 4).

[0073] Table 3. Reliability Verification of C22-1 Marking

[0074]

[0075] Table 4. Reliability Verification of C22-4 Marking

[0076]

[0077] Finally, the KASP molecular markers were applied to the screening of population progeny. The markers were used to detect drought-resistant materials in 120 materials from the K326 and Nanjiang 3 BC2 populations that survived rehydration after drought treatment. The results showed that the markers could effectively match the material phenotype with the genotype.

[0078] A total of 15 drought-resistant materials were tested using the C22-1 marker. Among them, 13 materials were homozygous and had the same genotype as the drought-resistant parent, Nanjiang 3, while 2 materials were heterozygous.

[0079] A total of 24 drought-resistant materials were tested using the C22-4 marker. Among them, 15 materials were homozygous and had the same genotype as the drought-resistant parent, Nanjiang 3, while the remaining 9 materials were heterozygous.

[0080] Therefore, both C22-1 and C22-4 can be used to identify drought-resistant genotypes in the K326 and Nanjiang 3 populations, such as Figure 3 ( Figure 3The results shown include, in addition to the plants used for verification, two homozygous parents (Tables 5 and 6).

[0081] Table 5. Application of C22-1 marking

[0082]

[0083] Table 6. Application of C22-4 marking

[0084]

[0085] IV. Conclusion:

[0086] Two KASP molecular markers related to drought resistance, C22-1 and C22-4, have been successfully developed. These markers exhibit high stability and accuracy and can be used in subsequent breeding programs for drought-resistant varieties. For example, tracking and analyzing the genotype of each individual plant in the constructed BC2 population can ensure the homozygosity of target gene regions and accurately determine the location and effect of drought-resistant genes. Furthermore, the combined application of the two markers yields more accurate results in drought-resistant variety breeding, providing more accurate scientific evidence for subsequent gene function research and breeding work, and accelerating the breeding process of drought-resistant tobacco varieties.

[0087] 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 SNP locus associated with drought tolerance in tobacco, characterized in that, Including C22-1 SNP sites and / or C22-4 SNP sites; The C22-1 SNP site is located at position 109518286 on the 22nd chromosome of tobacco, and its polymorphism is T / C. The C22-4 SNP site is located at position 110249618 on chromosome 22 of the tobacco plant, and its polymorphism is A / G.

2. A primer set targeting the SNP site of claim 1.

3. The primer set according to claim 2, characterized in that, The primer set targeting the C22-1 SNP site includes: a competitive specific forward primer 1 with the nucleotide sequence shown in SEQ ID NO:1, a competitive specific forward primer 2 with the nucleotide sequence shown in SEQ ID NO:2, and a universal reverse primer with the nucleotide sequence shown in SEQ ID NO:5; The primer set targeting the C22-4 SNP site includes: a competitive specific forward primer 1 with the nucleotide sequence shown in SEQ ID NO:6, a competitive specific forward primer 2 with the nucleotide sequence shown in SEQ ID NO:7, and a universal reverse primer with the nucleotide sequence shown in SEQ ID NO:

5.

4. The primer set according to claim 3, characterized in that, The 5' end of the competitive specific forward primer 1 and / or the competitive specific forward primer 2 is fluorescently labeled; The fluorescent labels of the competitive specific forward primer 1 and / or the competitive specific forward primer 2 are different.

5. The primer set of claim 4, wherein, The fluorescent labels include: FAM, HEX, ROX, Cy5 and / or JOE.

6. A reagent characterized in that, Contains at least one of a stabilizer, a solvent, and a preservative, and the primer set as described in any one of claims 2 to 5.

7. A kit for drought resistance tobacco variety selection, characterized in that, It includes the reagents described in claim 6, and reagents or devices for auxiliary detection.

8. The application of at least one of the following (I) to (IV) in the breeding of drought-resistant tobacco varieties: I) The SNP site as described in claim 1; II) The primer set according to any one of claims 2 to 5; III) The reagent as described in claim 6; IV) The kit according to claim 7.

9. A method for breeding drought resistant tobacco varieties, characterized in that, The tobacco is detected using the SNP site described in claim 1 as the target, or by using at least one of the methods shown in i) to iii) below, and then the drought resistance is determined based on the detection results: i) The primer set according to any one of claims 2 to 5; ii) The reagent as described in claim 6; iii) The kit according to claim 7.

10. The method of claim 9, wherein, The criteria for judging whether something is drought-resistant are as follows: If the C22-1 SNP site is homozygous C, then it indicates drought resistance; If the C22-1 SNP site is homozygous T, then it is drought-sensitive; If the C22-4 SNP site is homozygous for G, then it indicates drought resistance; If the C22-4 SNP site is homozygous A, then it is drought-sensitive.

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