A primer set for detecting SNP site of wheat TaWD40-4B.1 gene and application thereof

Abstract

This invention provides a primer set for detecting SNP sites in the wheat TaWD40-4B.1 gene. The SNP site is located at 1274 bp in the coding region of the wheat TaWD40-4B.1 gene, and the base type is G or A. The primer set includes upstream primers TaWD40-4B-F and TaWD40-4B-H, and a shared downstream primer TaWD40-4B-C. The method for detecting the genotype of the wheat TaWD40-4B.1 gene SNP site is as follows: using extracted genomic DNA from the wheat sample to be tested as a template, gradient PCR amplification is performed using upstream primers TaWD40-4B-F, TaWD40-4B-H, and the shared downstream primer TaWD40-4B-C. After completion, the genotype of the wheat TaWD40-4B.1 gene SNP site is determined using QuantStudio1. This invention also provides the application of the primer set described above for detecting SNP sites in the wheat TaWD40-4B.1 gene, used to prepare a kit for detecting the genotype of SNP sites in the wheat TaWD40-4B.1 gene. The primer set designed in this invention can specifically amplify the target fragment, avoiding non-specific reactions. Combined with KASP technology, it has high detection efficiency, can be directly associated with wheat drought resistance traits, accelerates the breeding of drought-resistant varieties, improves breeding efficiency, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of wheat SNP site technology, specifically relating to a primer set for detecting wheat TaWD40-4B.1 gene SNP sites and its application. Background Technology

[0002] A nonsense mutation at base 1274 of the coding region of TaWD40-4B.1 in a natural wheat population led to premature termination of the coding region, resulting in the full-length haplotype TaWD40-4B.1. G And truncated haplotype TaWD40-4B.1 A This nonsense mutation is highly associated with the drought resistance of natural wheat populations and carries the TaWD40-4B.1 base. G Wheat exhibits significantly higher drought resistance than wheat carrying TaWD40-4B.1. A wheat.

[0003] Therefore, the TaWD40-4B.1 gene has significant breeding value. However, current techniques for haplotype detection of TaWD40-4B.1 involve gene amplification followed by sequencing, a time-consuming, labor-intensive, and expensive method that is not suitable for high-throughput detection. Therefore, there is an urgent need to develop a convenient, efficient, and economical molecular marker detection method to better utilize the TaWD40-4B.1 gene in wheat drought-resistant molecular breeding. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a primer set for detecting SNP sites of the wheat TaWD40-4B.1 gene and its application. This primer set can specifically amplify the target fragment, avoid non-specific reactions, and ensure the accuracy of haplotype detection. Combined with KASP technology, it has high detection efficiency and short cycle, directly relates to wheat drought resistance traits, accelerates the breeding of drought-resistant varieties, improves breeding efficiency, and has good application prospects.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a primer set for detecting SNP sites of wheat TaWD40-4B.1 gene, wherein the nucleotide sequence of the coding region of wheat TaWD40-4B.1 gene is shown in SEQ ID No:1; the SNP site is located at 1274bp in the coding region of wheat TaWD40-4B.1 gene, and the base type is G or A; The primer set includes upstream primer TaWD40-4B-F, upstream primer TaWD40-4B-H, and a common downstream primer TaWD40-4B-C; The nucleotide sequence of the upstream primer TaWD40-4B-F is as follows: 5'-FAM-TCGACGGGTCCGTCAAGGTCTG-3'; The nucleotide sequence of the upstream primer TaWD40-4B-H is as follows: 5'-HEX-TCGACGGGTCCGTCAAGGTCTA-3'; The nucleotide sequence of the shared downstream primer TaWD40-4B-C is shown in SEQ ID No:2.

[0006] The present invention relates to a method for detecting the genotype of wheat TaWD40-4B.1 gene SNP sites using the primer set described above, characterized in that the method comprises: S1. Extract genomic DNA from the wheat sample to be tested; S2. Using the genomic DNA template of the wheat sample to be tested extracted in S1, gradient PCR amplification was performed using the upstream primers TaWD40-4B-F, TaWD40-4B-H, and the common downstream primer TaWD40-4B-C. S3. After gradient PCR amplification, the values ​​were read using QuantStudio1 to determine the genotype of the SNP site of the wheat TaWD40-4B.1 gene.

[0007] Preferably, the reaction system for gradient PCR amplification in S2 is: 5 μL HiGeno 2x Probe Mix A, 0.14 μL primer working solution, 1 μL DNA template, and 4 μL ddH2O; The reaction program for gradient PCR amplification is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing / extension at 61℃~55℃ for 40 s, wherein the gradient PCR at 61℃~55℃ decreases by 0.6℃ per cycle, for 10 cycles; denaturation at 95℃ for 20 s, annealing / extension at 55℃ for 40 s, for 30 cycles. The primer working solution includes: upstream primer TaWD40-4B-F, upstream primer TaWD40-4B-H, a common downstream primer TaWD40-4B-C, and ddH2O. The concentration of upstream primer TaWD40-4B-F is 12 μM, the concentration of upstream primer TaWD40-4B-H is 12 μM, and the concentration of the common downstream primer TaWD40-4B-C is 30 μM.

[0008] Preferably, the primer set for detecting the SNP site of the wheat TaWD40-4B.1 gene is used to prepare a kit for detecting the genotype of the SNP site of the wheat TaWD40-4B.1 gene.

[0009] Compared with the prior art, the present invention has the following advantages: 1. This invention designs a primer set for detecting the genotype of the wheat TaWD40-4B.1 gene SNP site based on the specific site of the TaWD40-4B.1 gene. The FAM and HEX marker primers target two key alleles respectively, which can specifically amplify the target fragment, avoid non-specific reactions, and ensure the accuracy of haplotype detection.

[0010] 2. This invention combines KASP technology, which has a short detection cycle and high efficiency, can meet the needs of large-scale population screening, is suitable for large-scale breeding applications, and improves breeding efficiency.

[0011] 3. This invention requires no complex equipment or operation, has a simple operation process, is easy to standardize and promote, and has broad application prospects.

[0012] 4. This invention is low in cost, high in cost-effectiveness, stable, highly reproducible, and provides reliable test results. It is directly related to the drought resistance trait of wheat and can be directly used for screening superior single plants and selecting parent lines, thereby accelerating the breeding of drought-resistant varieties.

[0013] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is a scatter plot of KASP classification in Embodiment 2 of the present invention. Detailed Implementation

[0015] Example 1

[0016] This example demonstrates the design of primer sets for detecting SNP sites in the wheat TaWD40-4B.1 gene.

[0017] Previous studies have found that the protein TaWD40, encoded by TraesCS4B02G072200.1 located on chromosome 4B, plays an important role in drought resistance, and its presence is known as TaWD40-4B.1. G (Drought-resistant) and TaWD40-4B.1 A(Sensitive) Two types of allelic variation. Because wheat is an allohexaploid crop, six allele information were found through the IWGSC website (https: / / www.wheatgenome.org / ): TraesCS4A02G242800.1 and TraesCS4A02G242900.1 on chromosome 4A, TraesCS4B02G072100.1 and TraesCS4B02G072300.1 on chromosome 4B, and TraesCS4D02G071000.1 and TraesCS4D02G071100.1 on chromosome 4D. The seven sequences were compared using DNAMAN software, revealing a similarity of over 89%. Based on this information, primers were designed. First, two genotyping primers were designed based on the SNP sites. FAM and HEX adapter sequences were added to the 5' ends of the designed upstream genotyping primers, respectively. The nucleotide sequence of the upstream primer TaWD40-4B-F is: 5'-FAM-TCGACGGGTCCGTCAAGGTCTG-3'; the nucleotide sequence of the upstream primer TaWD40-4B-H is: 5'-HEX-TCGACGGGTCCGTCAAGGTCTA-3'. Then, based on the sequence alignment results, specific sequences were selected for the design of the downstream common primer. The nucleotide sequence of the common downstream primer TaWD40-4B-C is shown in SEQ ID No:2. Example 2

[0018] This embodiment uses the primer set designed in Example 1 for the wheat TaWD40-4B.1 gene SNP site to detect the genotype of the wheat TaWD40-4B.1 gene SNP site.

[0019] Thirteen wheat varieties were selected: Jimai 26, Jimai 38, Shijiazhuang 10, Shimai 18, Shimai 25, Shimai 26, Shimai 28, Shimai 29, Shimai 30, Shimai 32, Shimai 33, Shimai 34, and Shimai 38. Five uniform and plump seeds were placed in a germination box, with a layer of filter paper underneath. The boxes were soaked in water in the dark for about 24 hours. After the seeds showed signs of sprouting, the germination boxes were transferred to a light incubator (20℃, 16h light / 8h darkness) and cultured for another 7 days. Leaves were then taken, and plant genomic DNA was extracted using a novel plant genomic DNA extraction kit (TIANGEN, DP320-03).

[0020] Using the extracted DNA as a template, gradient PCR amplification was performed using the primer set designed in Example 1. The primer working solution preparation is shown in Table 1, and the gradient PCR amplification system is shown in Table 2. HiGeno 2x Probe Mix A was purchased from Beijing Jiacheng Biotechnology Co., Ltd., and the gradient PCR amplification procedure is shown in Table 3.

[0021] Table 1 Primer working solution preparation Table 2 Detection System Table 3 Detection Procedure After the reaction, the values ​​were read using QuantStudio1, and the results are shown in Table 4. The SNP column represents the base at position 1274 for each variety. The genotyping scatter plot is shown below. Figure 1 As shown, the horizontal axis represents base A, and the vertical axis represents base G.

[0022] Table 4 Test Results Meanwhile, common primers WD40-F and WD40-R were designed, with nucleotide sequences shown in SEQ ID No: 3-4. Using the extracted DNA as a template, PCR amplification was performed. The PCR amplification reaction system is shown in Table 5. The 2x FidCycle Evo PCRMaster Mix premix was purchased from Shanghai Sangon Biotech Co., Ltd. The PCR amplification reaction procedure is shown in Table 6.

[0023] Table 5 PCR amplification reaction system Table 6 PCR Amplification Reaction Procedure The amplified product was then sent for sequencing, and the sequencing results were analyzed. The results are shown in Table 7. One column of the sequencing results represents the base at position 1274 of this variety.

[0024] Table 7 Sequencing results of PCR amplification products In summary, the gradient PCR amplification results and the PCR product amplification sequencing results are consistent, indicating that the detection results of this invention are reliable, and the designed primer set can be used to prepare a kit for detecting the SNP site genotype of the wheat TaWD40-4B.1 gene.

[0025] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A primer set for detecting SNP sites in the wheat TaWD40-4B.1 gene, characterized in that, The nucleotide sequence of the coding region of the wheat TaWD40-4B.1 gene is shown in SEQ ID No:1; the SNP site is located at 1274 bp in the coding region of the wheat TaWD40-4B.1 gene, and the base type is G or A; The primer set includes upstream primer TaWD40-4B-F, upstream primer TaWD40-4B-H, and a common downstream primer TaWD40-4B-C; The nucleotide sequence of the upstream primer TaWD40-4B-F is as follows: 5'-FAM-TCGACGGGTCCGTCAAGGTCTG-3'; The nucleotide sequence of the upstream primer TaWD40-4B-H is as follows: 5'-HEX-TCGACGGGTCCGTCAAGGTCTA-3'; The nucleotide sequence of the shared downstream primer TaWD40-4B-C is shown in SEQ ID No:

2.

2. A method for detecting the genotype of the wheat TaWD40-4B.1 gene SNP site using the primer set for detecting the wheat TaWD40-4B.1 gene SNP site as described in claim 1, characterized in that, The method is as follows: S1. Extract genomic DNA from the wheat sample to be tested; S2. Using the genomic DNA template of the wheat sample to be tested extracted in S1, gradient PCR amplification was performed using the upstream primer TaWD40-4B-F, upstream primer TaWD40-4B-H and the common downstream primer TaWD40-4B-C. S3. After gradient PCR amplification, the values ​​were read using QuantStudio1 to determine the genotype of the SNP site of the wheat TaWD40-4B.1 gene.

3. The method according to claim 2, characterized in that, The reaction system for gradient PCR amplification described in S2 is as follows: HiGeno 2x Probe Mix A 5 μL, primer working solution 0.14 μL, DNA template 1 μL, ddH2O 4 μL; The reaction program for gradient PCR amplification is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, annealing / extension at 61℃~55℃ for 40 s, wherein the gradient PCR at 61℃~55℃ decreases by 0.6℃ per cycle, for 10 cycles; denaturation at 95℃ for 20 s, annealing / extension at 55℃ for 40 s, for 30 cycles. The primer working solution includes: upstream primer TaWD40-4B-F, upstream primer TaWD40-4B-H, a common downstream primer TaWD40-4B-C, and ddH2O. The concentration of upstream primer TaWD40-4B-F is 12 μM, the concentration of upstream primer TaWD40-4B-H is 12 μM, and the concentration of the common downstream primer TaWD40-4B-C is 30 μM.

4. The application of a primer set as described in claim 1 for detecting SNP sites in the wheat TaWD40-4B.1 gene, characterized in that, The primer set for detecting the SNP site of the wheat TaWD40-4B.1 gene is used to prepare a kit for detecting the genotype of the SNP site of the wheat TaWD40-4B.1 gene.

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