dCAPS marker related to length of adventitious root under water stress in wheat and its application

Abstract

This invention relates to the fields of molecular biology and genetic breeding, and particularly to the dCAPS marker associated with the length of adventitious roots in wheat under waterlogging stress and its application. This dCAPS molecular marker is based on the 234,677,747th base of wheat chromosome 6B; the SNP base difference is either C or T. This molecular marker is accurate and efficient to detect, convenient and stable to amplify, and can be used for marker-assisted selection to improve the efficiency and accuracy of identifying the waterlogging stress resistance of wheat plants.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic breeding technology, and in particular to the dCAPS marker associated with the length of adventitious roots in wheat under waterlogging stress and its application. Background Technology

[0002] wheat( Triticum aestivum L. Wheat is one of the world's most important food crops, second only to corn and rice. It is widely cultivated and has a wide range of uses, primarily in food, animal feed, and beer brewing. Statistics show that waterlogging stress severely impacts approximately 16% of the world's arable land. As a dryland crop, wheat yields can decrease by 20-50% under waterlogging stress, and its impact on wheat production is far-reaching and cannot be ignored. Therefore, breeding waterlogging-resistant wheat varieties is an important breeding objective for ensuring food security.

[0003] Wheat resistance to waterlogging stress is a complex quantitative trait, formed by the combination of multiple traits, with root response being a major component. Waterlogging stress induces the growth and development of adventitious roots, thereby enhancing the oxygen supply capacity of root tissues. The resulting physiological and morphological changes can serve as an important indicator for evaluating wheat's waterlogging tolerance.

[0004] Traditional breeding methods rely on phenotypic screening for resistance identification, but these methods are time-consuming, have low throughput, and are easily affected by environmental factors, making them unsuitable for large-scale germplasm screening and efficient breeding. Molecular marker-assisted breeding provides an effective pathway for the precise screening of target traits. Among these, dCAPS markers, as a SNP-based functional marker technology, overcome enzyme restriction through primer mismatch design, offering advantages such as simple detection, controllable cost, and applicability to routine laboratories. However, there is currently a lack of dCAPS markers specifically developed for wheat waterlogging stress resistance, and existing marker systems struggle to achieve efficient and stable identification of waterlogging tolerance traits.

[0005] Therefore, using dCAPS molecular markers to accurately, rapidly, and easily identify wheat resistance to waterlogging stress is beneficial to accelerating the breeding process of waterlogging-resistant high-quality wheat and quickly selecting suitable waterlogging-resistant wheat germplasm for promotion to ensure food security. Summary of the Invention

[0006] In view of this, the present invention proposes a dCAPS marker related to the length of adventitious roots in wheat under waterlogging stress and its application. This molecular marker, s_6B_234677747_dCAPS, can be used to screen wheat materials with different waterlogging stress resistance levels, and can also be used in the field of wheat genetics and breeding to cultivate new wheat varieties with different waterlogging stress resistance levels.

[0007] The specific technical solution is as follows: In a first aspect, the present invention provides a molecular marker associated with wheat waterlogging stress resistance, said molecular marker being located at a single nucleotide polymorphism (SNP) site on the wheat genome; The SNP site corresponds to position 132 bp in the genome sequence shown in SEQ ID NO.4; The SNP site is located at position 234677747 on the wheat chromosome; and there is a C / T base difference at this physical position.

[0008] Furthermore, the molecular marker is located on wheat chromosome 6B; Furthermore, the full-sequence version of the wheat reference genome is IWGSC RefSeqv1.0.

[0009] Furthermore, the waterlogging stress resistance is characterized by the adventitious root length of wheat after waterlogging stress.

[0010] Furthermore, the primer pair sequences used to amplify the molecular marker are as follows: Upstream primer F: 5'-ATGGTTGTTGCACGCCATTG-3'; Downstream primer R: 5'-TGCTTTGCTCCTGCCATGCCCGCGAGCTTTGCATGTGCTA-3'; the amplification product is 172bp, and its sequence is shown in SEQ ID NO.3. The molecular marker site is located at 132bp of the amplification product fragment.

[0011] Furthermore, the introduced mismatched base site is located at 136 bp of the amplified product fragment.

[0012] In a second aspect, the present invention provides a primer pair for amplifying the above-mentioned molecular marker, the primer pair sequence being as follows: Upstream primer F: 5'-ATGGTTGTTGCACGCCATTG-3'; Downstream primer R: 5'-TGCTTTGCTCCTGCCATGCCCGCGAGCTTTGCATGTGCTA-3'.

[0013] In a third aspect, the present invention provides a detection kit comprising the aforementioned primer pairs.

[0014] The present invention provides, in a fourth aspect, the use of the above-described molecular marker, or the above-described primer pair, or the above-described detection kit in any of the following: (1) Identification, breeding and improvement of wheat resistance to waterlogging stress; (2) Early prediction of wheat resistance to waterlogging stress; (3) Marker-assisted breeding of wheat; (4) Screening or breeding high-yield wheat.

[0015] The present invention provides a method for identifying whether wheat has resistance to waterlogging stress in a fifth aspect, comprising the following steps: (1) Extract genomic DNA from wheat plants; (2) Using the genomic DNA described in step (1) as a template, perform PCR amplification using the primer pair; (3) Use restriction endonucleases to perform genotyping and analysis of the test samples based on electrophoretic bands; If the base at the SNP site is of type C, the enzyme digestion is successful, and the wheat plant being tested is a material resistant to high waterlogging stress; if the base at the SNP site is of type T, the enzyme digestion fails, and the wheat plant being tested is a material resistant to low waterlogging stress.

[0016] Furthermore, the PCR amplification reaction system is as follows: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL of each of 10 μmol / L primers, 1 μL of 100 ng / μL template DNA, 9.5 μL of sterile water, and a total reaction volume of 25 μL.

[0017] Furthermore, the PCR reaction procedure is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0018] Furthermore, the restriction endonucleases include: NheI-HF restriction endonuclease, AluI restriction endonuclease, and BfaI restriction endonuclease.

[0019] Furthermore, the restriction endonuclease is preferably an NheI-HF restriction endonuclease.

[0020] Furthermore, the recognition sequence of the NheI-HF restriction endonuclease is G / CTAGC.

[0021] Furthermore, the band size of the successfully digested enzyme product is 135 bp; the band size of the unsuccessfully digested enzyme product is 172 bp.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a dCAPS molecular marker based on SNP sites significantly associated with waterlogging stress resistance in wheat plants. This marker offers accurate and efficient detection, convenient and stable amplification, and can be used for marker-assisted selection to improve the efficiency and accuracy of identifying waterlogging stress resistance in wheat plants. This will facilitate the acceleration of breeding processes for waterlogging-resistant, high-quality wheat, and the rapid selection of suitable waterlogging-resistant wheat germplasm for widespread application, ensuring food security. Attached Figure Description

[0023] Figure 1 (a) in the figure shows the electrophoresis results of the molecular marker enzyme digestion experiment; Figure 1 (b) in the diagram shows the violin diagram of the adventitious root lengths for different genotypes. Detailed Implementation

[0024] The molecular markers and applications provided by this invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0025] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0026] The wheat material used in this invention can be obtained from the Institute of Crop Sciences, Zhejiang University.

[0027] All biochemical reagents used in this invention are commercially available.

[0028] The product sequence of the amplified molecular marker described in this invention corresponds to that shown in SEQ ID NO.3, namely: The molecular marker is located at 132 bp in this sequence, exhibiting a C / T base difference.

[0029] The genomic sequence of the molecular marker described in this invention corresponds to that shown in SEQ ID NO.4, namely: The molecular marker is located at 132 bp in this sequence, exhibiting a C / T base difference.

[0030] Example 1 (1) Trial supply of materials 355 wheat germplasm resources were selected and planted in Hangzhou in 2025, and waterlogging stress was applied at the three-leaf-one-heart stage.

[0031] (2) Characteristic determination On the 4th day after waterlogging stress, the length of all adventitious roots of a single wheat plant was measured manually, and the total length of all adventitious roots of a single wheat plant was calculated.

[0032] (3) GWAS analysis and SNP molecular marker determination Combining the measured adventitious root lengths and the 304,744 SNP markers in this population, a GWAS analysis was performed using the efficient mixed association model EMMAx. The results showed that a single SNP marker located on chromosome 6B was significantly associated with the total adventitious root length after waterlogging stress. The difference in total adventitious root length caused by this SNP is shown in the figure below. Figure 1 As shown in (b), the total length of adventitious roots of the CC type wheat genotype was significantly greater than that of the TT type wheat genotype after waterlogging stress. This SNP is located at the 234677747th base of chromosome 6B. This SNP site has a C / T difference. The genomic sequence of this SNP site is shown in SEQ ID NO.4.

[0033] Example 2 (1) Trial supply of materials Ten wheat samples were selected for analysis of the target region of s_6B_234677747_dCAPS. Under normal conditions, the wheat plants were planted until tillering, then subjected to waterlogging stress with tap water. After four days of stress, the lengths of all adventitious roots were measured for each wheat plant, and the total length of all adventitious roots for each plant was calculated. The details are shown in Table 1, including six samples with high total adventitious root lengths and four samples with low total adventitious root lengths.

[0034] Table 1. Total length of adventitious roots of 10 wheat germplasm materials after waterlogging stress Group wheat germplasm SNP Total length of indeterminate roots (cm) Enzyme digestion status 1 79TK098-516 TT 2.20 Uncut 2 TJK04-3 TT 6.00 Uncut 3 6319 TT 10.93 Uncut 4 79TK112-1067B TT 13.50 Uncut 5 IWA8604165 CC 46.73 Cut 6 Hulutou CC 50.47 Cut 7 455 CC 50.50 Cut 8 168 CC 55.05 Cut 9 Gandom CC 55.43 Cut 10 86PK1306-002.00 CC 56.45 Cut (2) Obtaining SNP markers Based on the SNP site information and the wheat whole genome sequence information, dCAPS marker primers were developed. The upstream primer F is 5'-ATGGTTGTTGCACGCCATTG-3' (SEQ ID NO.1); the downstream primer R is 5'-TGCTTTGCTCCTGCCATGCCCGCGAGCTTTGCATGTGCTA-3' (SEQ ID NO.2). The amplified size is 172 bp, and the SNP site is located at 132 bp of the amplified fragment, as shown in SEQ ID NO.3. Using the above primer pairs, the variation at position 234677747 of wheat chromosome 6B was detected to detect the length of adventitious roots after waterlogging stress in wheat.

[0035] (3) DNA extraction DNA was extracted from fresh leaves during the seedling stage using the CTAB method. The detailed steps are as follows: A) Take about 3-5 g of tender wheat leaves, add CTAB preheated at 65℃ and grind, then bathe in a 65℃ water bath for 1 hour, shaking gently 3-5 times during the process. B) Add an equal volume of chloroform:isoamyl alcohol (24:1) solution and shake well; centrifuge at 10,000 rpm for 5 min, and transfer the supernatant into a new centrifuge tube; C) Add 2 / 3 volume of ice-cold isopropanol, shake well, and let stand at -20℃ for 20 min to precipitate DNA; D) Centrifuge at 10,000 rpm for 5 min, discard the supernatant; wash once each with 75% and 100% ethanol, air dry, and dissolve in distilled water. Store at -20℃ until use.

[0036] (4) PCR The PCR amplification reaction system was as follows: 12.5 μL of 2 × Rapid Taq Master Mix (Vazyme), 1 μL of 10 μmol / L Primer, 1 μL of 100 ng / μL template DNA, 9.5 μL of sterile water, and a total reaction volume of 25 μL.

[0037] The PCR reaction was performed on a PCR instrument. The reaction program included: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0038] (5) Restriction endonuclease digestion Ten test materials were digested using NheI-HF restriction endonuclease. The digestion system consisted of: 0.4 μL NheI-HF, 5 μL PCR product, 1 μL rCutSmart™ Buffer, and 3.6 μL sterile water. Genotyping of the ten test materials was performed using SNP markers, and the results are as follows: Figure 1 As shown in (a). Among them, the SNP typing is divided into two groups: the CC type has two bands (172bp and 135bp), and the TT type has only one band (172bp).

Claims

1. Molecular markers associated with wheat resistance to waterlogging stress, characterized in that, The molecular marker is located at a single nucleotide polymorphism (SNP) site on the wheat genome; The SNP site corresponds to position 132 bp in the genome sequence shown in SEQ ID NO.4; The SNP site is located at position 234677747 on the wheat chromosome; and there is a C / T base difference at this physical position.

2. The molecular marker associated with wheat waterlogging stress as described in claim 1, characterized in that, The molecular marker is located on wheat chromosome 6B; The full-sequence version of the wheat reference genome is IWGSC RefSeqv1.

0.

3. The molecular marker associated with waterlogging stress resistance in wheat as claimed in claim 1, wherein, The waterlogging stress resistance is characterized by the length of adventitious roots in wheat under waterlogging stress.

4. The molecular marker associated with wheat waterlogging stress as described in claim 1, characterized in that, The primer pair sequences used to amplify the molecular marker are as follows: Upstream primer F: 5'-ATGGTTGTTGCACGCCATTG-3'; Downstream primer R: 5'-TGCTTTGCTCCTGCCATGCCCGCGAGCTTTGCATGTGCTA-3'; The amplification product is 172 bp, and its sequence is shown in SEQ ID NO.

3. The molecular marker site is located at 132 bp of the amplification product fragment.

5. A primer pair for amplifying the molecular marker according to any one of claims 1 to 3, characterized in that, The primer pair sequences are as follows: Upstream primer F: 5'-ATGGTTGTTGCACGCCATTG-3'; Downstream primer R: 5'-TGCTTTGCTCCTGCCATGCCCGCGAGCTTTGCATGTGCTA-3'.

6. A test kit characterized in that, The kit contains the primer pair as described in claim 5.

7. The use of the molecular marker as described in any one of claims 1 to 4, or the primer pair as described in claim 5, or the detection kit as described in claim 6, in any of the following: (1) Identification, breeding and improvement of wheat resistance to waterlogging stress; (2) Early prediction of wheat resistance to waterlogging stress; (3) Marker-assisted breeding of wheat; (4) Screening or breeding high-yield wheat.

8. A method for identifying whether wheat possesses resistance to waterlogging stress, characterized in that, Includes the following steps: (1) Extract genomic DNA from wheat plants; (2) Using the genomic DNA described in step (1) as a template, perform PCR amplification using the primer pair; (3) Use restriction endonucleases to perform genotyping and analysis of the test samples based on electrophoretic bands; If the base at the SNP site is of type C, then the enzyme digestion is successful, and the wheat plant to be tested is a material resistant to high waterlogging stress. If the base at the SNP site is of type T, the enzyme digestion will fail, and the wheat plant to be tested is a low-waterlogging stress resistant material. The restriction endonucleases include: NheI-HF restriction endonuclease, AluI restriction endonuclease, and BfaI restriction endonuclease.

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