TaTGAL6-2A molecular marker and application thereof in detection of drought resistance of wheat

Abstract

The invention discloses a TaTGAL6-2A molecular marker and application of the TaTGAL6-2A molecular marker in detection of drought resistance of wheat, and belongs to the technical field of biology. The technical problem to be solved by the invention is how to detect the drought resistance of wheat. The TaTGAL6-2A molecular marker disclosed by the invention is nucleotide, corresponding to the 4124 site of SEQ ID No.1 in a sequence table, in a wheat genome, and is G or A. Experiments prove that the drought resistance of the homozygous wheat with the nucleotide A at the 4124 site corresponding to the SEQ ID No.1 in the sequence table in the genome is greater than that of the homozygous wheat with the nucleotide A at the site G. By detecting the molecular marker, the drought-resistant wheat can be quickly and accurately found. The invention provides a new method for wheat molecular marker-assisted selective breeding, and has important significance in culture or research of stress-resistant wheat varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the TaTGAL6-2A molecular marker and its application in detecting wheat drought resistance. Background Technology

[0002] With the development of molecular biology, molecular marker-assisted breeding has provided a convenient and rapid method for selecting target traits in wheat. Utilizing molecular markers to discover and utilize superior gene resources provides a foundation for improving breeding efficiency and offers an efficient pathway for crop 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 molecular markers of drought resistance in wheat in the detection or auxiliary detection of wheat drought resistance;

[0005] The wheat drought resistance molecular marker is the nucleotide in the wheat genome corresponding to position 4124 of SEQ ID No. 1 in the sequence listing, which is either G or A.

[0006] Specifically, the substance used to detect wheat molecular markers is a primer pair named TaTGAL6-2A-Primer or a set of reagents for detecting wheat molecular markers;

[0007] The TaTGAL6-2A-Primer consists of two single-stranded DNA sequences shown in SEQ ID No. 4 and SEQ ID No. 5 of the sequence listing;

[0008] The kit for detecting the wheat molecular marker includes a primer pair named TaTGAL6-2A-Primer-Sa1I, which consists of two single-stranded DNA molecules as shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing.

[0009] Furthermore, the kit also includes the restriction endonuclease Sa1I.

[0010] The complete set of reagents may be TaTGAL6-2A-Primer-Sa1I, or may be composed of TaTGAL6-2A-Primer-Sa1I and Sa1I.

[0011] Furthermore, the drought resistance of homozygous wheat whose genome corresponds to nucleotide A at position 4124 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 4124 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 drought resistance molecular markers and determining wheat drought resistance according to the following method:

[0013] The drought resistance of homozygous wheat whose genome corresponds to nucleotide A at position 4124 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 4124 of SEQ ID No. 1 in the sequence listing.

[0014] Specifically, the detection of the wheat drought-resistant molecular markers is performed using the substance used to detect wheat drought-resistant molecular markers.

[0015] The substance used to detect drought-resistant molecular markers in wheat is also within the scope of protection of this invention.

[0016] The wheat drought-resistant molecular markers mentioned above are also within the scope of protection of this invention.

[0017] This invention also provides any of the following applications:

[0018] Application of the wheat drought-resistant molecular markers described in X1) in the breeding of drought-resistant wheat;

[0019] X2) The application of the wheat drought resistance molecular markers in detecting or assisting in the detection of wheat drought resistance;

[0020] X3) The application of the substances used to detect drought-resistant molecular markers in wheat in the breeding of drought-resistant wheat;

[0021] X4) The application of the substance used to detect drought-resistant molecular markers in wheat in the preparation and cultivation of drought-resistant wheat products;

[0022] X5) The application of the substance described above for detecting wheat drought resistance molecular markers in the preparation of products for detecting or assisting in the detection of wheat drought resistance;

[0023] The application of the detection or auxiliary detection method for wheat drought resistance described in X6) in the breeding of drought-resistant wheat;

[0024] X7) The application of detecting the substance in the wheat genome corresponding to nucleotide 4124 of SEQ ID No. 1 in the sequence listing in the breeding of drought-resistant wheat;

[0025] X8) The application of detecting the substance in the wheat genome corresponding to nucleotide 4124 of SEQ ID No. 1 in the sequence listing in the preparation of drought-resistant wheat products.

[0026] The present invention also provides a method for breeding drought-resistant wheat, the method comprising: detecting the 4124th nucleotide in the wheat genome corresponding to SEQ ID No.1 in the sequence listing, and selecting wheat whose nucleotide at the 4124th nucleotide in the wheat genome corresponding to SEQ ID No.1 in the sequence listing is A as a parent for breeding.

[0027] In this invention, the drought resistance can be reflected in the root growth of wheat under drought conditions.

[0028] In this invention, the wheat can be any of the 32 wheat materials or any of the wheat materials in Table 1 or their descendants; the 32 wheat materials 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.

[0029] Experiments have shown that homozygous wheat with nucleotide A at position 4124 of SEQ ID No. 1 in the genome exhibits greater drought resistance than homozygous wheat with nucleotide G at the same position. By detecting the wheat molecular markers of this invention, drought-resistant wheat can be identified rapidly and accurately. This invention provides a novel method for marker-assisted selection breeding of wheat, and is of great significance in the cultivation of stress-resistant wheat varieties or in related research. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is a partial detection result of the molecular markers of this invention. The electrophoretic bands in lane G are 196 bp and 25 bp (the 25 bp band is too small to be shown), the electrophoretic bands in lane A are 221 bp, and lane M represents the DNA molecular weight standard.

[0032] Figure 2 This is a statistical result of the root length drought resistance coefficient of different homozygous types of the TaTGAL6-2A gene in a natural wheat population. Detailed Implementation

[0033] 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.

[0034] 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 commercially available.

[0035] In the following examples, unless otherwise specified, 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.

[0036] Example 1: Obtaining molecular markers related to drought resistance in wheat

[0037] I. Obtaining the TaTGAL6-2A gene polymorphism site

[0038] 1. Based on the characteristics of the wheat TaTGAL6-2A genomic DNA sequence, specific primers were designed for its genome. The primer sequences are as follows:

[0039] TaTGAL6-2A-Primer-F (forward primer): 5′-CCTAGTACCCACGCACGTCC-3′ (SEQ ID No. 4 in the sequence listing);

[0040] TaTGAL6-2A-Primer-R (reverse primer): 5′-ATGTATTTACACATCCTCGATCCCT-3′ (SEQ ID No. 5 in the sequence listing).

[0041] The recognition sequences of TaTGAL6-2A-Primer-F and TaTGAL6-2A-Primer-R are located in the second intron and downstream of the TaTGAL6-2A gene, respectively.

[0042] 2. Using genomic DNA from 32 wheat materials (all from the National Germplasm Bank) as templates, PCR amplification was performed using the primers from step 1 to obtain PCR amplification products. The obtained PCR amplification products were then sequenced and their sequences were compared.

[0043] 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.

[0044] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 45 s, for 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0045] Sequence analysis revealed a G and A polymorphism (R represents G or A) at position 4124 of the TaTGAL6-2A genomic DNA (SEQ ID No. 1), which was denoted as the molecular marker TaTGAL6-2A-4124.

[0046] The names of the 32 wheat materials used 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, and White Rough Wheat.

[0047] II. Detection of Molecular Markers

[0048] 1. For the molecular marker at position 4124 of the TaTGAL6-2A gene shown in SEQ ID No. 1 of the sequence listing, design primers to detect this marker. The primer sequences are as follows:

[0049] TaTGAL6-2A-Primer-Sa1I-F (forward primer): 5′-GTGGCTGGCACGCAGATC-3′ (SEQ ID No. 2);

[0050] TaTGAL6-2A-Primer-Sa1I-R (forward primer): 5′-CCGACGAGGCCCTTGAGCTTGTCGA-3′ (SEQ ID No. 3).

[0051] 2. The steps for detecting the molecular markers of the wheat sample using the primers from step 1 are as follows:

[0052] Using the wheat genomic DNA to be tested as a template, PCR amplification was performed using the primers from step 1, yielding PCR amplification product A. The nucleotide sequence of PCR amplification product A is from position 3929 to 4149 of SEQ ID No. 1.

[0053] 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.

[0054] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 20 s, for 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0055] The PCR amplification product A obtained in step 1 was digested with the restriction endonuclease Sa1I to obtain digested product B. The electrophoretic detection results of part of the digested product B are as follows: Figure 1 As shown. If enzyme digestion product B contains two DNA fragments of 196 bp and 25 bp in size and does not contain a DNA fragment of 221 bp, it indicates that the nucleotide corresponding to position 4124 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA being tested is G, and the wheat being tested is homozygous. If enzyme digestion product B contains one DNA fragment of 221 bp in size and does not contain the DNA fragments of 196 bp and 25 bp, it indicates that the nucleotide corresponding to position 4124 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA being tested is A, and the wheat being tested is homozygous. If enzyme digestion product B contains three DNA fragments of 196 bp, 25 bp, and 221 bp in size, it indicates that the nucleotides corresponding to position 4124 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA being tested are G and A, and the wheat being tested is heterozygous.

[0056] Example 2: Correlation analysis between wheat molecular markers and drought resistance

[0057] The molecular markers from Example 1 were used to genotype the natural population (Table 1), and the correlation between the molecular markers and drought resistance was analyzed. The specific steps are as follows:

[0058] 1. Detection of molecular markers

[0059] In a natural population of 323 hexaploid wheat samples, each wheat variety was selected as the test wheat and its molecular markers were detected according to step two of Example 1. The nucleotide corresponding to position 4124 of SEQ ID No. 1 in the sequence listing was determined for each individual wheat sample. All wheat varieties in the natural population were sourced from the National Germplasm Resource Bank.

[0060] The detection results of the molecular markers are shown in Table 1. "A" indicates a homozygous form where the nucleotide at position 4124 of SEQ ID No. 1 in the sequence listing is A; "G" indicates a homozygous form where the nucleotide at position 4124 of SEQ ID No. 1 in the sequence listing is G; and "-" indicates a heterozygous form where the nucleotide at position 4124 of SEQ ID No. 1 in the sequence listing is both A and G. Then, the genomic DNA of each wheat variety was amplified and sequenced using the primer pairs TaTGAL6-2A-Primer-Sa1I-F and TaTGAL6-2A-Primer-Sa1I-R from Example 1. The amplification system and conditions were the same as in Example 1. The results showed that the sequences of the PCR products of each wheat variety were positions 3929 to 4149 of SEQ ID No. 1.

[0061] Table 1. Detection results of various molecular markers in wheat from natural populations

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] 2. Correlation analysis between molecular markers and drought resistance

[0068] Seeds from natural populations of different wheat varieties were placed in 60mm×90mm petri dishes. After water treatment for 1 day, they were transplanted into 96-well black plastic boxes sterilized with 1% hydrogen peroxide. Two treatment conditions were set up: PEG treatment and water treatment. Each black plastic box had 12 wells and 8 rows, with 12 seeds per row. There were 3 replicates. After 3 days of treatment under different conditions, the root length of different wheat varieties was measured, and the average root length drought resistance coefficient of each replicate was calculated.

[0069] The PEG treatment involved placing the germinated wheat in a 20% (by mass) PEG aqueous solution; the control involved placing the germinated wheat in water.

[0070] Root length drought resistance coefficient = root length under PEG treatment / root length under control.

[0071] The molecular marker (i.e., the nucleotide at position 4124 of SEQ ID No. 1) in Example 1 was correlated with the root length drought resistance coefficient using the GLM model in Tassel 5.0 software.

[0072] The statistical results of drought resistance coefficients for various wheat root lengths are shown in Table 2 and... Figure 2 The root length drought resistance coefficient of homozygous wheat with position A at SEQ ID No. 1 in the genome is significantly greater than that of homozygous wheat with position G at the same site. This indicates that the molecular markers of the present invention are related to wheat drought resistance and can be used to breed drought-resistant wheat varieties.

[0073] Table 2. Root length drought resistance coefficients of different homozygous wheat varieties in natural populations.

[0074]

[0075] In Table 2, * indicates a p-value < 0.05 for significance analysis.

[0076] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of substances that detect molecular markers of drought resistance in wheat in the detection or auxiliary detection of wheat drought resistance; The wheat drought resistance molecular marker is the nucleotide in the wheat genome corresponding to position 4124 of SEQ ID No. 1 in the sequence listing, which is either G or A.

2. The application according to claim 1, characterized in that: The substance used to detect wheat molecular markers is a primer pair named TaTGAL6-2A-Primer or a set of reagents for detecting wheat molecular markers. The TaTGAL6-2A-Primer consists of two single-stranded DNA sequences shown in SEQ ID No. 4 and SEQ ID No. 5 of the sequence listing; The kit for detecting the wheat molecular marker includes a primer pair named TaTGAL6-2A-Primer-Sa1I, which consists of two single-stranded DNA molecules as shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing.

3. The application according to claim 2, characterized in that: The kit also includes the restriction endonuclease Sa1I.

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 4124 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 4124 of SEQ ID No. 1 in the sequence listing.

5. A method for detecting or assisting in the detection of wheat drought resistance, characterized in that: The method includes detecting the wheat drought-resistant molecular markers described in claim 1, and determining wheat drought resistance according to the following method: The drought resistance of wheat whose genome corresponds to nucleotide A at position 4124 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 4124 of SEQ ID No. 1 in the sequence listing.

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

7. The substance for detecting wheat drought resistance molecular markers as described in any of claims 1-3.

8. The wheat drought-resistant molecular marker as described in claim 1.

9. Any of the following applications: X1) The application of the wheat drought-resistant molecular markers as described in claim 1 in the breeding of drought-resistant wheat; X2) The application of the wheat drought-resistant molecular markers described in claim 1 in the detection or auxiliary detection of wheat drought resistance; X3) The application of any of the substances described in claims 1-3 for detecting drought-resistant molecular markers in wheat in the breeding of drought-resistant wheat; X4) The use of any of the substances described in claims 1-3 for detecting drought-resistant molecular markers in wheat in the preparation of drought-resistant wheat products; X5) The use of any of the substances for detecting wheat drought resistance 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 of claim 5 or 6 in the cultivation of drought-resistant wheat; X7) The application of detecting the substance in the wheat genome corresponding to nucleotide 4124 of SEQ ID No. 1 in the sequence listing in the breeding of drought-resistant wheat; X8) The application of detecting the substance in the wheat genome corresponding to nucleotide 4124 of SEQ ID No. 1 in the sequence listing in the preparation of drought-resistant wheat products.

10. Methods for cultivating drought-resistant wheat, including: Nucleotide 4124 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing was detected, and wheat with nucleotide A corresponding to SEQ ID No. 1 in the sequence listing was selected as the parent for breeding.

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