Molecular markers and methods for identifying drought resistance in wheat seedlings

Abstract

This invention discloses molecular markers and methods for identifying drought resistance in wheat seedlings. The molecular markers for identifying drought resistance in wheat seedlings disclosed in this invention are nucleotides in the wheat genome corresponding to position 1038 of SEQ ID No. 1 in the sequence listing, which are either A or G. Experiments have shown that this molecular marker is associated with drought resistance in wheat; homozygous wheat with nucleotide A at position 1038 of SEQ ID No. 1 in the genome has greater drought resistance than homozygous wheat with nucleotide G at position 1038 of SEQ ID No. 1 in the genome. Using the molecular markers of this invention, wheat varieties with high drought resistance in the seedling stage can be quickly and accurately identified. This invention provides a new method for marker-assisted selection breeding of wheat and has significant implications for the cultivation of drought-resistant, high-yielding wheat varieties or for related research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically molecular markers and methods for identifying drought resistance in wheat seedlings. Background Technology

[0002] Wheat is one of my country's three major staple crops, and high and stable wheat yields are crucial for ensuring national food security. Drought and water shortages severely impact wheat production, making it essential to improve wheat's drought resistance as a key means to guarantee high and stable yields. With the development of molecular biology, molecular marker-assisted breeding provides a convenient and rapid method for selecting target traits. Discovering superior gene resources and developing molecular markers lays the foundation for efficient genetic improvement and germplasm innovation. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to detect the drought resistance of wheat.

[0004] To address the aforementioned technical problems, this invention first provides the application of substances that detect wheat molecular markers in the detection or auxiliary detection of wheat drought resistance;

[0005] The wheat molecular marker is the nucleotide in the wheat genome corresponding to position 1038 of SEQ ID No. 1 in the sequence listing, which is either A or G.

[0006] In the above applications, the substance used to detect wheat molecular markers may contain a primer pair named TaNRT2-Primer-BsmAI or a primer pair named TaNRT2-6A-Primer.

[0007] The TaNRT2-Primer-BsmAI consists of two single-stranded DNA sequences shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing;

[0008] The TaNRT2-6A-Primer consists of two single-stranded DNA sequences shown in SEQ ID No. 4 and SEQ ID No. 5 of the sequence listing.

[0009] In the above applications, the kit may also include the restriction endonuclease BsmAI.

[0010] The complete reagent kit may consist solely of TaNRT2-Primer-BsmAI or TaNRT2-6A-Primer, or it may be composed of TaNRT2-Primer-BsmAI and the restriction endonuclease BsmAI.

[0011] In the above applications, the drought resistance of homozygous wheat whose genome corresponds to nucleotide A at position 1038 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than that of homozygous wheat whose genome corresponds to nucleotide G at position 1038 of SEQ ID No. 1 in the sequence listing.

[0012] This invention also provides a method for detecting or assisting in the detection of wheat drought resistance, the method comprising detecting the wheat molecular markers and determining the drought resistance of wheat according to the following method:

[0013] The drought resistance of homozygous wheat whose genome corresponds to nucleotide A at position 1038 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than that of homozygous wheat whose genome corresponds to nucleotide G at position 1038 of SEQ ID No. 1 in the sequence listing.

[0014] In the above method, the detection of the wheat molecular marker can be performed using the substance for detecting wheat molecular markers.

[0015] In the above method, detecting the wheat molecular marker using the TaNRT2-6A-Primer may include: performing PCR amplification on wheat genomic DNA using the TaNRT2-6A-Primer to obtain an amplification product; sequencing the amplification product to determine the nucleotide in the wheat genome corresponding to position 1038 of SEQ ID No. 1 in the sequence listing.

[0016] The PCR amplification reaction system can be as follows: 12.2 μL ddH2O, 4.0 μL 5×PCR buffer, 0.4 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 1.6 μL dNTPs (2.5 mmol / L), 0.4 μL transfastpfu enzyme (5 U), and 1 μL template DNA (20 ng / μL). Both the 5×PCR buffer and transfastpfu enzyme (5 U) are products of Beijing TransGen Biotech Co., Ltd.

[0017] The PCR amplification reaction conditions can be: 95℃ for 2 min; 95℃ for 50 s, 53℃ for 50 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0018] The above method, in which the detection of the wheat molecular marker using the TaNRT2-Primer-BsmAI, may include: performing PCR amplification on wheat genomic DNA using the TaNRT2-Primer-BsmAI to obtain an amplification product; sequencing the amplification product to determine the nucleotide in the wheat genome corresponding to position 1038 of SEQ ID No. 1 in the sequence listing.

[0019] The PCR amplification reaction system can be as follows: 3.6 μL ddH2O, 0.2 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 5 μL 2×PCR Mix, and 1 μL template DNA (20 ng / μL). The 2×PCR Mix is ​​a product of Zhuangmeng Biotechnology Co., Ltd., catalog number ZT201A.

[0020] The PCR amplification reaction conditions can be: 95℃ for 2 min; 95℃ for 50 s, 58℃ for 50 s, 72℃ for 30 s, 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0021] The above method, using the TaNRT2-Primer-BsmAI and BsmAI to detect the wheat molecular marker, may include: performing PCR amplification of wheat genomic DNA using the TaNRT2-Primer-BsmAI to obtain an amplification product; digesting the amplification product with BsmAI to obtain a digestion product; detecting the size of the digestion product; and determining the nucleotide corresponding to position 1038 of SEQ ID No. 1 in the wheat genome according to the following method:

[0022] If the enzyme digestion product contains a DNA fragment of 299 bp and does not contain DNA fragments of 275 bp and 24 bp, then the nucleotide at position 1038 of SEQ ID No. 1 in the wheat genomic DNA is A;

[0023] If the enzyme digestion product contains two DNA fragments of 275bp and 24bp in size and does not contain a DNA fragment of 299bp in size, then the nucleotide at position 1038 of SEQ ID No. 1 in the wheat genomic DNA is G;

[0024] If the enzyme digestion product contains three DNA fragments of sizes 299bp, 275bp, and 24bp, then the nucleotides corresponding to position 1038 of SEQ ID No. 1 in the wheat genomic DNA are A and G (i.e., A at this position on one chromosome and G at this position on another chromosome).

[0025] The size of the enzyme digestion products can be determined by electrophoresis or sequencing.

[0026] The substance used to detect wheat molecular markers is also within the scope of protection of this invention.

[0027] The wheat molecular markers mentioned above are also within the scope of protection of this invention.

[0028] This invention also provides any of the following applications:

[0029] The application of wheat molecular markers described in X1) in wheat breeding;

[0030] X2) The application of wheat molecular markers in detecting or assisting in the detection of wheat drought resistance;

[0031] X3) The application of the substances used to detect wheat molecular markers in wheat breeding;

[0032] X4) The application of the substance for detecting wheat molecular markers in the preparation of wheat breeding products;

[0033] X5) The application of the substances described for detecting wheat molecular markers in the preparation of products for detecting or assisting in the detection of wheat drought resistance;

[0034] The application of the methods described in X6 for detecting or assisting in the detection of wheat drought resistance in wheat breeding;

[0035] X7) The application of detecting substances in the wheat genome corresponding to nucleotide 1038 of SEQ ID No. 1 in the sequence listing in the selection of wheat with excellent drought resistance;

[0036] X8) The application of detecting substances in the wheat genome corresponding to nucleotide 1038 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent drought resistance.

[0037] The present invention also provides a wheat breeding method, the method comprising: detecting the nucleotide corresponding to the 1038th nucleotide of SEQ ID No. 1 in the wheat genome, and selecting wheat whose nucleotide corresponding to the 1038th nucleotide of SEQ ID No. 1 in the wheat genome is A as the parent for breeding.

[0038] In this invention, the drought resistance can be seedling-stage drought resistance. In one embodiment of this invention, the drought resistance is the drought resistance of wheat at the three-leaf stage.

[0039] In this invention, the wheat can be any of the 32 wheat varieties and any wheat material in Table 1 or its descendants. The 32 wheat varieties are PANDAS, An85zhong124-1, Yanzhan 1, Bawangbian, Beijing 10, Beijing 14, Cangzhou Wheat, Changwu 131, Chang 6878, Dali 1, DanR8093, Fengkang 13, Jimai 41, Jimai 6, Jin 2148-7, Jinghe 8922, Linkang 5108, Baiqi Wheat, Changle 5, Hongheshang, Beijing 8686, 04-044, 04-030, Chun 22 9th-25, Zigan Baimangxian, Jingpin 10, Chun 04 9th-5-1, Chun 45 9th-50-1, Neixiang 188, Jing 411, China Spring, and White Rough Wheat.

[0040] This invention, through genetic variation analysis of the TaNRT2-6A gene in a natural wheat population, identified a single SNP located at position 1038 of SEQ ID No. 1, which is both A and G. Association analysis and natural population validation confirmed that this SNP is associated with drought resistance in wheat. Homozygous wheat with nucleotide A at position 1038 of SEQ ID No. 1 exhibits greater drought resistance than homozygous wheat with nucleotide G at position 1038 of SEQ ID No. 1. Experiments demonstrated that detecting the nucleotide at position 1038 of SEQ ID No. 1 in the genome can rapidly and accurately identify wheat varieties with high drought resistance at the seedling stage. This invention provides a novel method for marker-assisted selection breeding of wheat, and is of significant importance in the cultivation of drought-resistant, high-yielding wheat varieties or in related research.

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0042] Figure 1 This is a partial detection result of the molecular markers used in this invention. Based on the CAPS-1038 molecular marker designed at site 1038, the electrophoretic band size in lane A (SNP-A) is 299 bp, and the band sizes in lanes G (SNP-G) are 275 bp and 24 bp, respectively. Due to the long electrophoresis time, the 24 bp fragment is somewhat blurry. M: 100 bp marker. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are all commercially available. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0044] Example 1: Obtaining the polymorphic site of the drought resistance-related gene TaNRT2-6A

[0045] I. Obtaining the TaNRT2-6A gene polymorphic site

[0046] (1) Based on the characteristics of the wheat TaNRT2-6A genomic DNA sequence, specific primers for the genome were designed. The primer sequences are as follows:

[0047] TaNRT2-6A-Primer-F (forward primer): 5′-TCGCTGCCATGCTTAGTTTT-3′ (SEQ ID No. 4 in the sequence listing);

[0048] TaNRT2-6A-Primer-R (reverse primer): 5′-CCTTGCTTGATCGAGCTAGG-3′ (SEQ ID No. 5 in the sequence listing).

[0049] The recognition sequences of TaNRT2-6A-Primer-F and TaNRT2-6A-Primer-R are located upstream and downstream of the TaNRT2-6A gene promoter, respectively.

[0050] (2) Using the genomic DNA of 32 wheat materials (all from the National Germplasm Resource Bank) as templates, PCR amplification was performed using the primers in step (1) to obtain PCR amplification products. Then, the obtained PCR amplification products were sequenced and sequence aligned.

[0051] The PCR amplification system used (20 μL) consisted of: 12.2 μL ddH2O, 4.0 μL 5×PCR buffer, 0.4 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 1.6 μL dNTPs (2.5 mmol / L), 0.4 μL transfastpfu enzyme (5 U), and 1 μL template DNA (20 ng / μL). Both the 5×PCR buffer and transfastpfu enzyme (5 U) were products of Beijing TransGen Biotech Co., Ltd.

[0052] The PCR amplification conditions were: 95℃ for 2 min; 95℃ for 50 s, 53℃ for 50 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0053] Sequence analysis revealed an A and G polymorphism at position 1038 of the TaNRT2-6A genomic DNA (SEQ ID No. 1). In SEQ ID No. 1, R represents A or G.

[0054] The names of these 32 wheat materials are as follows: PANDAS, An85zhong124-1, Yanzhan No.1, Bawangbian, Beijing No.10, Beijing No.14, Cangzhou Wheat, Changwu 131, Chang 6878, Dali No.1, DanR8093, Fengkang 13, Jimai 41, Jimai No.6, Jin 2148-7, Jinghe 8922, Linkang 5108, Baiqi Wheat, Changle No.5, Hongheshang, Beijing 8686, 04-044, 04-030, Chun 229th-25, Zigan Baimangxian, Jingpin No.10, Chun 04 9th-5-1, Chun 45 9th-50-1, Neixiang 188, Jing 411, China Spring, White Rough Wheat.

[0055] II. Acquisition of Molecular Markers and Their Application in Genotyping

[0056] 1. Obtaining molecular markers

[0057] The SNP site at position 1038 of the TaNRT2-6A gene shown in SEQ ID No. 1 of the sequence listing is designated as CAPS-1038. Primers capable of detecting each marker are designed as follows:

[0058] TaNRT2-Primer-BsmAI-F (forward primer): 5′-ACACTTGCATGTGTAGGTGGC-3′ (SEQ ID No. 2);

[0059] TaNRT2-Primer-BsmAI-R (reverse primer): 5′-TGGATCAATGAGATATATATGTTGAGAGA-3′ (SEQ ID No. 3).

[0060] 2. Identification of wheat genotypes

[0061] (1) When the wheat to be tested is a wheat with a homozygous TaNRT2-6A gene, the steps for identifying the genotype of the wheat to be tested using molecular markers and primers in step 1 are as follows:

[0062] Using wheat genomic DNA as a template, PCR amplification was performed using the CAPS-1038-labeled primers from step 1, yielding PCR amplification product A. The nucleotide sequence of PCR amplification product A is positions 769 to 1067 of SEQ ID No. 1.

[0063] The PCR amplification system (10 μL) consisted of: 3.6 μL ddH2O, 0.2 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 5 μL 2×PCR Mix, and 1 μL template DNA (20 ng / μL). The 2×PCR Mix was a product of Zhuangmeng Biotechnology Co., Ltd., catalog number ZT201A.

[0064] The PCR amplification conditions were: 95℃ for 2 min; 95℃ for 50 s, 58℃ for 50 s, 72℃ for 30 s, for 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0065] (2) The PCR amplification product A obtained in step (1) was digested with the restriction endonuclease BsmAI to obtain digested product B. The electrophoretic detection results of part of the digested product B are as follows: Figure 1 If the enzyme digestion product B is only 299 bp, it indicates that the nucleotide corresponding to position 1038 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA to be tested is A, and the wheat to be tested is homozygous. The genotype of the wheat to be tested is recorded as SNP-A. If the enzyme digestion product B is 275 bp and 24 bp, it indicates that the nucleotide corresponding to position 1038 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA to be tested is G, and the wheat to be tested is homozygous. The genotype of the wheat to be tested is recorded as SNP-G. If the enzyme digestion product B consists of three DNA fragments of sizes 299 bp, 275 bp and 24 bp, it indicates that the nucleotides corresponding to position 1038 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA to be tested are A and G, and the wheat to be tested is heterozygous. The haplotype of the wheat to be tested is recorded as SNP-AG.

[0066] Therefore, the following method can be used to genotype the wheat to be tested and determine its genotype:

[0067] 1) Extract genomic DNA from the wheat to be tested;

[0068] 2) Using the genomic DNA from step 1) as a template, PCR amplification was performed using primers TaNRT2-Primer-BsmAI to obtain PCR amplification product A.

[0069] 3) Digest PCR amplification product A with BsmAI to obtain digested product B;

[0070] The size of enzyme digestion product B can be detected (by electrophoresis and sequencing). If enzyme digestion product B contains only a band of 299 bp, then the genotype of the wheat being tested is SNP-A.

[0071] If enzyme digestion product B contains only bands of 275bp and 24bp, then the genotype of the wheat being tested is SNP-G.

[0072] Example 2: Association analysis between wheat genotype and seedling survival rate under repeated drought conditions

[0073] I. Genotyping of Natural Population 1 and its Association with Seedling Survival Rate During Repeated Droughts

[0074] Genotyping of natural population 1 (Table 2) was performed using the CAPS-1038 marker from Example 1, and association analysis was conducted between genotype and seedling survival rate during repeated droughts. The specific steps are as follows:

[0075] 1. Genotyping

[0076] In a natural population 1 consisting of 323 hexaploid wheat accessions, each wheat variety was used as a test wheat and its genotype was determined according to the method described in Example 1. All wheat varieties in the natural population 1 were sourced from the National Germplasm Resource Bank.

[0077] The genotyping results are shown in Table 1. All wheat varieties were homozygous, and the nucleotide sequence of PCR amplification product A was from position 769 to 1067 of SEQ ID No. 1. Then, the genomic DNA of each wheat variety was amplified using the primer pairs TaNRT2-6A-Primer-F and TaNRT2-6A-Primer-R from Example 1. The amplification system and conditions were the same as in Example 1. The results showed that the PCR product sequence of each wheat variety was SEQ ID No. 1 in the sequence listing, and that each wheat variety had an SNP at position 1038, i.e., A and G polymorphisms. This indicates that the CAPS-1038 marker of the present invention can be used to detect wheat varieties with genotypes SNP-A and SNP-G.

[0078] Table 1. Statistical results of wheat genotypes in natural population 1

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] 2. Association analysis between genotype and seedling survival rate under drought treatment

[0085] Wheat natural population 1 was planted under a dry shed at the headquarters of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. When the wheat reached the three-leaf stage, water was withheld for drought treatment, and the soil moisture content was about 22.0%. After 10 days of drought treatment, the soil moisture content dropped to 4.3%, and the soil was re-watered for 5 days. Then, a second drought treatment was carried out, and after 15 days, the soil moisture content dropped to 3.9%, and the soil was re-watered for 5 days. Then, a third drought treatment was carried out, which lasted for 15 days, and the soil moisture content dropped to 2.3%. Five days after the soil was re-watered, the number of seedlings was investigated.

[0086] The survival rates of the two genotypes and the seedling drought treatment were analyzed using the GLM model with Tassel 5.0 software.

[0087] Table 2 shows the association analysis results between the CAPS-1038 marker (the nucleotide at position 1038 of SEQ ID No. 1 in the sequence listing) formed by natural population 1 and the survival rate of seedlings under drought treatment. The CAPS-1038 marker was significantly associated with the survival rate of drought treatment. The survival rate of wheat with position A at position 1038 of SEQ ID No. 1 in the genome (i.e., wheat homozygous for A at this position) was significantly higher than that of wheat with position G at this position (i.e., wheat homozygous for G at this position), indicating that this marker can be used to breed wheat varieties with strong drought resistance.

[0088] Table 3 shows the statistical results of drought treatment survival rates for two wheat genotypes formed in natural population 1. The drought treatment survival rate of wheat genotype SNP-A was significantly higher than that of genotype SNP-G. This indicates that the genotype of this invention is correlated with the survival rate of wheat seedlings under drought treatment, and can be used to breed wheat varieties with strong drought resistance.

[0089] Table 2. Results of association analysis between TaNRT2-6A marker and seedling drought treatment survival rate in wheat natural population 1

[0090] drought treatment survival rate P-value Survival rate of the third drought treatment 0.02077

[0091] Table 3. Statistical results of drought treatment survival rates of the two TaNRT2-6A genotypes in wheat natural population 1.

[0092]

[0093] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a substance that detects wheat molecular markers in detecting or assisting in the detection of wheat drought resistance; wherein the wheat molecular marker is a nucleotide in the wheat genome corresponding to position 1038 of SEQ ID No. 1 in the sequence listing, and is A or G.

2. The application according to claim 1, characterized in that: The substance used to detect wheat molecular markers contains a primer pair named TaNRT2-Primer-BsmAI or a primer pair named TaNRT2-6A-Primer. The TaNRT2-Primer-BsmAI consists of two single-stranded DNA sequences shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing; The TaNRT2-6A-Primer consists of two single-stranded DNA sequences shown in SEQ ID No. 4 and SEQ ID No. 5 of the sequence listing.

3. The application according to claim 2, characterized in that: The substance also includes the restriction endonuclease BsmAI.

4. The application according to any one of claims 1-3, characterized in that: The drought resistance of wheat whose genome corresponds to nucleotide A at position 1038 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than that of wheat whose genome corresponds to nucleotide G at position 1038 of SEQ ID No. 1 in the sequence listing.

5. A method for detecting or assisting in the detection of wheat drought resistance, comprising detecting the wheat molecular markers described in claim 1, and determining the drought resistance of wheat according to the following method: the drought resistance of wheat whose nucleotide position A at position 1038 of SEQ ID No. 1 in the genome is greater than or candidate greater than the drought resistance of wheat whose nucleotide position G at position 1038 of SEQ ID No. 1 in the genome is greater than or candidate greater than the drought resistance of wheat whose nucleotide position G at position 1038 of SEQ ID No. 1 in the genome is greater than or candidate greater than the drought resistance of wheat.

6. The method according to claim 5, characterized in that: The detection of the wheat molecular markers described in claim 1 is performed using any of the substances described in claims 1-3 for detecting wheat molecular markers.

7. Any of the following applications: X1) The application of wheat molecular markers as described in claim 1 in wheat drought resistance breeding; X2) The application of the wheat molecular markers described in claim 1 in the detection or auxiliary detection of wheat drought resistance; X3) The application of the substance for detecting wheat molecular markers as described in any of claims 1-3 in wheat drought resistance breeding; X4) The use of any of the substances for detecting wheat molecular markers as described in claims 1-3 in the preparation of wheat drought-resistant breeding products; X5) The use of any of the substances for detecting wheat molecular markers as described in claims 1-3 in the preparation of products for detecting or assisting in the detection of wheat drought resistance; X6) The application of the method according to claim 5 or 6 in wheat drought resistance breeding; X7) The application of detecting the substance in the wheat genome corresponding to nucleotide 1038 of SEQ ID No. 1 in the sequence listing in the breeding of wheat with excellent drought resistance; X8) The application of detecting substances in the wheat genome corresponding to nucleotide 1038 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent drought resistance.

8. Methods for breeding wheat to improve drought resistance, including: Nucleotide 1038 of SEQ ID No. 1 in the wheat genome was detected, and wheat with nucleotide A corresponding to SEQ ID No. 1 in the wheat genome was selected as the parent for breeding.

Images