Method for auxiliary screening of resistant soybeans and special primer thereof

Abstract

The invention relates to the technical field of gene engineering, and discloses a method for auxiliary screening of saline-alkaline resistant soybeans and a special primer thereof. According to the method, whether the soybean variety has resistance or not is determined through genome DNA extraction, resistance gene fragment PCR amplification, gel electrophoresis and sequencing analysis. Specifically, a special primer designed for an SNP site and an Indel site in a soybean genome is used for PCR amplification, for the SNP site, when the 105th site of the sequence of SEQ ID NO: 3 is A, it is indicated that soybean has resistance, and for the Indel site, a forward primer SEQ ID NO: 1 and a reverse primer SEQ ID NO: 2 are used for amplification. The method provides a simple, rapid and high-precision means for screening out saline-alkaline tolerant soybean germplasm resources, and has important significance for improving soybean varieties to adapt to saline-alkaline soil.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and more specifically to a method for assisting in screening resistant soybeans and a special primer thereof. Background Art

[0002] In modern agricultural production, plant resistance has always been one of the important goals of breeding work. It enables plants to maintain normal growth and development through their own physiological, biochemical and genetic mechanisms when facing various adverse environmental conditions. This ability is of great significance for improving crop yield and quality, enhancing its adaptability and ensuring food security. Resistance can be divided into many types, including disease resistance, insect resistance, drought resistance, cold resistance and salt-alkali resistance. However, the problem of soil salinization is becoming increasingly serious and has become the main factor restricting the sustainable development of agriculture. About 30% of the world's arable land is affected by salinization to varying degrees. In the process of salt-alkali stress, the soybean root system, as a medium for direct communication with the soil environment, is the first to be affected. Long-term exposure to saline-alkali soil will damage the soybean cell membrane due to ion poisoning, which will seriously affect its absorption of water and nutrients. At the same time, endogenous hormone synthesis is disordered, root growth vitality is lost, and eventually the aboveground leaves gradually die. Salt-intolerant varieties will even wither and die, making it urgent to cultivate salt-alkali tolerant soybean varieties.

[0003] The traditional screening method of soybean salt-alkali tolerant varieties mainly relies on field planting observation and phenotypic identification. This method is like judging the health of its underground root system only by observing the branches and leaves of trees exposed to the ground, ignoring the comprehensive impact of the complex environment inside the soil on the plant. It not only consumes a lot of time, manpower and material resources, but is also easily disturbed by environmental factors, and has low accuracy and efficiency. With the rise of genetic engineering technology, molecular marker methods have been gradually applied to the screening of soybean salt-alkali tolerant varieties. However, most of the molecular markers selected by the existing methods are not closely related to the soybean salt-alkali tolerant gene, and it is difficult to accurately point to the key resistance site, resulting in a significant reduction in the reliability of the screening results. The primers designed at the same time lack specificity, and are prone to produce non-specific amplification products during PCR amplification, which interfere with subsequent detection and analysis and increase the complexity and error of the experiment. Therefore, the present invention provides a method for auxiliary screening of resistant soybeans and special primers thereof to solve the above-mentioned technical problems. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a simple, fast and high-precision means for screening out salt-alkali tolerant soybean germplasm resources, which is of great significance for improving soybean varieties to adapt to saline-alkali soils.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for auxiliary screening of resistant soybeans and its special primers, wherein the screening method comprises the following working steps:

[0006] S1, extracting soybean genomic DNA;

[0007] S2, using the genomic DNA as a template, performing PCR amplification using a primer combination targeting a SNP site in the salt-alkali resistance gene and a primer combination targeting an Indel site;

[0008] S3. Perform sequencing analysis on the amplified products of the SNP sites, and combine the gel electrophoresis results of the amplified products of the Indel sites to comprehensively determine whether the soybean is a salt- and alkali-tolerant variety;

[0009] The SNP site is the 105th nucleotide from the 5' end of SEQ ID NO:3. When the site is A, it indicates that the soybean has salt-alkali resistance. The amplification product of the Indel site determines resistance by whether there is an insertion or deletion of more than 10 bp.

[0010] Preferably, the dedicated primers are designed based on specific SNP sites and Indel sites in soybean resistance genes.

[0011] Preferably, the SNP site is located at position 105 of SEQ ID NO: 3, and its association with salt-alkali resistance is verified through salt-alkali stress experiments.

[0012] Preferably, the total volume of the PCR amplification reaction system is 20-30 μL, including 0.5-1.5 μL soybean genomic DNA, 2-3 μL 10×Buffer, 0.4-0.6 μL dNTPs, 0.4-0.6 μL each of forward and reverse primers, 0.1-0.3 μL TaqDNA polymerase, and the balance is deionized water.

[0013] Preferably, the PCR amplification steps are pre-denaturation at 94-96°C for 3-5 min, denaturation at 90-95°C for 20-40 s, annealing at 50-60°C for 20-40 s, extension at 70-75°C for 30-60 s, the number of cycles is 25-35 times, and finally extension at 70-75°C for 5-10 min.

[0014] Preferably, the Indel site has no insertion or deletion in the resistant variety and has a deletion of more than 10 bp in the susceptible variety, including the forward primer SEQ ID NO: 15'-AGCTGACCTAGGTGAAGCA-3' and the reverse primer SEQ ID NO: 25'-TTCGGTGATGCCATTAGTTC-3'

[0015] Preferably, the SNP site is the 105th nucleotide of SEQ ID NO: 3, and when it is A, it is judged as a resistant variety, including the forward primer SEQ ID NO: 45'-TGGACCTTGAAGTCTGCTGA-3' and the reverse primer SEQ ID NO: 55'-CCTGGAAGTCTGCTGAGTCT-3'.

[0016] In summary, the present invention has the following beneficial effects:

[0017] 1. The present invention accurately screens out salt-alkali tolerant soybean germplasm resources. Breeders can use these excellent resources as parents and use traditional breeding techniques or modern biotechnology to perform hybridization, breeding and other operations, and gradually integrate salt-alkali tolerant genes into more soybean varieties, which is helpful to cultivate new soybean varieties that are adapted to saline-alkali soil environments, expand the planting range of soybeans, and improve the yield and quality of soybeans in saline-alkali land. This is of great significance for alleviating the global reduction in soybean planting area and yield decline caused by soil salinization, thereby ensuring global food security.

[0018] 2. The present invention only needs to extract soybean genomic DNA, perform PCR amplification using special primers, detect the amplified product using gel electrophoresis technology, and sequence the amplified product, which simplifies the experimental process, reduces the requirements for the technical level of operators, shortens the screening cycle, and can obtain reliable screening results in a shorter time. At the same time, by directly designing special primers for specific SNP sites and Indel sites closely related to soybean resistance genes, the primers have strong specificity, can accurately amplify target gene fragments related to salt and alkali resistance, avoid non-specific amplification interference, and greatly improve the accuracy of screening. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] 1. Experimental Materials

[0021] Unless otherwise specified, the materials, reagents, etc. used in the examples and comparative examples of the present invention can be obtained from commercial sources.

[0022] DNA extraction kit: purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product model DP305.

[0023] Taq PCR Master Mix: purchased from Takara Biotechnology (Dalian) Co., Ltd., catalog number RR820A, provides the buffer system, dNTPs and Taq DNA polymerase required for PCR reaction.

[0024] Primers: forward primer SEQ ID NO: 1, reverse primer SEQ ID NO: 2, forward primer SNP-F, reverse primer SNP-R, all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0025] 2. Acquisition and verification of SNP sites and Indel sites

[0026] In order to identify gene loci associated with salt-alkali tolerance, soybean genome data was first obtained through whole genome sequencing or targeted sequencing technology. The specific steps are as follows:

[0027] Whole genome sequencing or targeted sequencing: Soybean samples were sequenced using the Illumina HiSeq series of high-throughput sequencing platforms to generate high-quality sequence reads.

[0028] Sequence alignment and variant detection: The sequencing reads were aligned with the reference genome using the Burrows-Wheeler Aligner software. Subsequently, the Genome Analysis Toolkit was used for variant detection to identify single nucleotide polymorphisms (SNPs) and insertion / deletion (Indel) sites.

[0029] Selection of SNP and Indel sites: Based on literature reports and previous experimental results, SNP sites and Indel sites closely related to salt-alkali resistance were selected.

[0030] 3. PCR amplification and gel electrophoresis detection

[0031] After obtaining the candidate SNP and Indel sites, PCR amplification is performed to confirm the existence and status of these sites. The specific steps are as follows:

[0032] Construction of PCR reaction system: A 25 μL reaction system was constructed in a PCR tube. The specific components were: 1 μL soybean genomic DNA, 12.5 μL 2×Taq PCR Master Mix, 10 μM forward primer SEQ ID NO: 1 0.5 μL, 10 μM reverse primer SEQ ID NO: 2 0.5 μL, 10 μM forward primer SNP-F 0.5 μL, 10 μM reverse primer SNP-R 0.5 μL, and the balance was deionized water.

[0033] PCR amplification conditions: amplification was performed in a PCR instrument, with pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, 35 cycles, and finally extension at 72°C for 10 min to obtain the amplified product.

[0034] Gel electrophoresis detection: The amplified products were separated by 1.5% agarose gel electrophoresis, and a UV transilluminator was used to detect whether amplified fragments of the expected size were present.

[0035] 4. Sequencing and Data Analysis

[0036] To further confirm the status of SNP and Indel sites, high-throughput sequencing of the amplified products is required. The specific steps are as follows:

[0037] Sequencing service: The amplified product will be sent to Shanghai Bioengineering Co., Ltd. for Sanger sequencing.

[0038] Data analysis: The sequencing results were compared with the reference sequence using the sequence analysis software BioEdit to confirm whether there was a specific base mutation at the SNP site (for example, when the 105th base of SEQ ID NO:3 was A, it indicated that the soybean had salt-alkali resistance), and whether there was an insertion or deletion variation at the Indel site (for example, there was a deletion of more than 10 bp).

[0039] 5. Performance Testing

[0040] To verify the effectiveness of the screening method, the following performance tests were performed:

[0041] Example 1

[0042] The screening method of this embodiment includes the following steps:

[0043] S1. Select three soybean seedlings of different varieties, numbered S1-S3, which come from non-saline-alkali land environments, to preliminarily verify the effectiveness of the screening method. According to the operating instructions of the Tiangen DP305 DNA extraction kit, extract genomic DNA from the leaves of the three soybean seedlings, measure the concentration of the extracted DNA using a spectrophotometer, adjust it to 50 ng / μL, and store it in a -20℃ refrigerator for later use;

[0044] S2. Construct a 25 μL reaction system in a PCR tube, with the following components: 1 μL soybean genomic DNA, 12.5 μL 2×Taq PCR Master Mix, 10 μM forward primer SEQ ID NO: 1 0.5 μL, 10 μM reverse primer SEQ ID NO: 2 0.5 μL, 10 μM forward primer SNP-F 0.5 μL, 10 μM reverse primer SNP-R 0.5 μL, and the remainder is deionized water;

[0045] Amplification was performed in a PCR instrument, with 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s, 35 cycles, and finally 72°C extension for 10 min to obtain the amplified product;

[0046] S3. The amplified products were separated by 1.5% agarose gel electrophoresis, detected by UV transilluminator, and sent to Shanghai Biotech Co., Ltd. for sequencing.

[0047] Example 2

[0048] The screening method of this embodiment includes the following steps:

[0049] S1. Select soybean seeds collected from saline-alkali land and sow them in a greenhouse. When the seedlings grow to two true leaves, select 3 plants, numbered Y1-Y3. These soybean seedlings come from saline-alkali land environments and are used to evaluate the performance of soybean varieties in the actual saline-alkali environment. According to the operating instructions of the Tiangen DP305 DNA extraction kit, extract genomic DNA from the leaves of the 3 soybean seedlings, respectively. Use a spectrophotometer to measure the concentration of the extracted DNA, adjust it to 60 ng / μL, and store it in a -20°C refrigerator for later use.

[0050] S2. Construct a 30 μL reaction system in a PCR tube, with the following components: 1.5 μL soybean genomic DNA, 15 μL 2×Taq PCR Master Mix, 10 μM forward primer SEQ ID NO: 1 0.6 μL, 10 μM reverse primer SEQ ID NO: 2 0.6 μL, 10 μM forward primer SNP-F 0.6 μL, 10 μM reverse primer SNP-R 0.6 μL, and the remainder is deionized water;

[0051] Amplification was performed in a PCR instrument, with 96°C pre-denaturation for 4 min, 93°C denaturation for 25 s, 58°C annealing for 25 s, 73°C extension for 50 s, 38 cycles, and finally 73°C extension for 8 min to obtain the amplified product;

[0052] S3. The amplified products were separated by 2% agarose gel electrophoresis, detected by UV transilluminator, and sent to Shanghai Biotech Co., Ltd. for sequencing.

[0053] Example 3

[0054] The screening method of this embodiment includes the following steps:

[0055] S1. Select soybean varieties with known salt-alkali tolerance as positive control (CK+), and soybean varieties with known salt-alkali intolerant performance as negative control (CK-). At the same time, select 3 soybean seedlings to be tested, numbered T1-T3. These soybean seedlings are from varieties with unknown salt-alkali resistance, which are used to further verify the effectiveness and reliability of the screening method. According to the operating instructions of Tiangen DP305 DNA extraction kit, extract genomic DNA from the leaves of the 3 soybean seedlings respectively, use a spectrophotometer to measure the concentration of the extracted DNA, adjust it to 40ng / μL, and store it in a -20℃ refrigerator for later use;

[0056] S2. Construct a 22 μL reaction system in a PCR tube, the specific components of which are: 0.8 μL soybean genomic DNA, 11 μL 2×Taq PCR Master Mix, 10 μM forward primer SEQ ID NO: 1 0.4 μL, 10 μM reverse primer SEQ ID NO: 2 0.4 μL, 10 μM forward primer SNP-F 0.4 μL, 10 μM reverse primer SNP-R 0.4 μL, and the balance is deionized water;

[0057] Amplification was performed in a PCR instrument, with pre-denaturation at 94°C for 3 min, followed by denaturation at 92°C for 40 s, annealing at 52°C for 40 s, and extension at 70°C for 60 s, for 40 cycles, and finally extension at 70°C for 5 min to obtain the amplified product;

[0058] S3. The amplified products were separated by 1.8% agarose gel electrophoresis, detected by UV transilluminator, and sent to Shanghai Biotech Co., Ltd. for sequencing.

[0059] Performance Testing

[0060] After the experimental operation of Examples 1-3, the following test results were obtained, and the test results are shown in Table 1.

[0061] Table 1

[0062]

[0063]

[0064] From the results shown in Table 1 above, it can be seen that the screening of soybean resistance by designing special primers directly for specific SNP sites and Indel sites closely related to soybean resistance genes has important application value in the process of soybean molecular marker-assisted breeding.

[0065] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for assisting in screening resistant soybeans, characterized in that: The resistance is salt and alkali resistance, which includes the following working steps: S1, extracting soybean genomic DNA; S2, using the genomic DNA as a template, performing PCR amplification using a primer combination targeting a SNP site in the salt-alkali resistance gene and a primer combination targeting an Indel site; S3. Perform sequencing analysis on the amplified products of the SNP sites, and combine the gel electrophoresis results of the amplified products of the Indel sites to comprehensively determine whether the soybean is a salt- and alkali-tolerant variety; The SNP site is the 105th nucleotide from the 5' end of SEQ ID NO:

3. When the site is A, it indicates that the soybean has salt-alkali resistance. The amplification product of the Indel site determines resistance by whether there is an insertion or deletion of more than 10 bp.

2. A special primer used in the method for assisting in screening resistant soybeans according to claim 1, characterized in that: The dedicated primers are designed based on specific SNP sites and Indel sites in soybean resistance genes.

3. The special primer according to claim 2, characterized in that The SNP site is located at position 105 of SEQ ID NO: 3, and its association with salt-alkali resistance was verified through salt-alkali stress experiments.

4. The special primer according to claim 2, characterized in that: The total volume of the PCR amplification reaction system is 20-30 μL, including 0.5-1.5 μL soybean genomic DNA, 2-3 μL 10×Buffer, 0.4-0.6 μL dNTPs, 0.4-0.6 μL each of forward and reverse primers, 0.1-0.3 μL Taq DNA polymerase, and the balance is deionized water.

5. The special primer according to claim 2, characterized in that The PCR amplification steps are pre-denaturation at 94-96°C for 3-5 minutes, denaturation at 90-95°C for 20-40 seconds, annealing at 50-60°C for 20-40 seconds, extension at 70-75°C for 30-60 seconds, the number of cycles is 25-35 times, and finally extension at 70-75°C for 5-10 minutes.

6. The special primer according to claim 2, characterized in that The amplified product of the Indel site has no insertion or deletion in the resistant variety, and has a deletion of more than 10 bp in the susceptible variety, including the forward primer SEQ ID NO: 1 5'-AGCTGACCTAGGTGAAGCA-3' and the reverse primer SEQ ID NO: 2 5'-TTCGGTGATGCCATTAGTTC-3'.

7. The special primer according to claim 2, characterized in that The SNP site is the 105th nucleotide of SEQ ID NO:

3. When it is A, it is determined to be a resistant variety, including the forward primer SEQ ID NO: 4 5'-TGGACCTTGAAGTCTGCTGA-3' and the reverse primer SEQ ID NO: 5 5'-CCTGGAAGTCTGCTGAGTCT-3'.

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