SNP marker on long arm of wheat chromosome 5a associated with cold resistance and application thereof
By detecting SNP molecular markers on the long arm of wheat chromosome 5A, the problem of rapidly identifying or comparing wheat cold resistance has been solved, enabling early breeding selection and improving breeding efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2024-12-05
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies make it difficult to quickly and easily predict or compare the cold resistance of wheat, resulting in slow progress in breeding.
This invention provides a method and kit for detecting SNP molecular markers (deoxyribonucleotide at position 457 of SEQ ID NO: 1 is G or A) on the long arm of wheat chromosome 5A. Through techniques such as DNA sequencing and restriction enzyme fragment length polymorphism, it can assist in the identification or comparison of cold resistance in wheat.
It enables rapid and accurate identification or comparison of cold resistance in the early stages of wheat development, significantly shortening the breeding selection cycle and improving breeding efficiency.
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Abstract
Description
Technical Field
[0001] This application relates to the fields of biotechnology and molecular breeding, specifically to a SNP marker on the long arm of wheat chromosome 5A that is associated with cold resistance and its application. Background Technology
[0002] Common wheat is one of the world's three major staple crops, providing approximately one-fifth of the global population's total calories. Its high and stable yields directly impact the living standards of the Chinese people and national food security. Low-temperature damage has long been a natural factor leading to severe wheat yield reductions. In recent years, due to the continuous occurrence of low-temperature damage, wheat yields have been severely reduced, making the improvement of wheat's cold resistance a focus of attention for breeders and geneticists.
[0003] However, genetic improvement of wheat cold resistance has progressed slowly, mainly because cold resistance is a complex quantitative trait, and field phenotypic identification is relatively difficult. Molecular marker-assisted selection breeding technology, which uses molecular markers controlling target trait loci / gene linkages and then performs genotyping through molecular biology detection, can achieve the goal of selecting for the target trait, unaffected by environmental factors, and rapidly and efficiently breed target resource materials. Summary of the Invention
[0004] The technical problem this application aims to solve is: how to quickly and easily predict or compare the cold resistance of wheat, for example, how to predict, assist in predicting, compare, and / or assist in comparing the cold resistance of wheat in the early stages of wheat development (seedling stage). To solve this technical problem, this application provides the following technical solution:
[0005] This application provides the use of substances for detecting SNP molecular markers in any of the following:
[0006] A1) Application in identifying or assisting in the identification of wheat cold resistance;
[0007] A2) Application in the preparation of products for identifying or assisting in the identification of wheat cold resistance;
[0008] A3) Application in comparing or assisting in comparing the cold resistance of wheat;
[0009] A4) Application in the preparation of products for comparing or assisting in comparing the cold resistance of wheat;
[0010] A5) Applications in plant breeding;
[0011] A6) Application in the preparation of plant breeding products;
[0012] The SNP molecular marker is the 457th deoxyribonucleotide of SEQ ID NO: 1 (in the 5' to 3' orientation), which is G or A.
[0013] In the above applications, the substance can be a substance that detects the SNP molecular marker by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP microarray. The SNP microarray includes microarrays based on nucleic acid hybridization reactions, microarrays based on single-base extension reactions, microarrays based on allele-specific primer extension reactions, microarrays based on one-step reactions, microarrays based on primer ligation reactions, microarrays based on restriction endonuclease reactions, microarrays based on protein-DNA binding reactions, and microarrays based on fluorescent molecule-DNA binding reactions.
[0014] Furthermore, in the application described, the substance contains PCR primers for amplifying wheat genomic DNA fragments including the SNP molecular marker.
[0015] Furthermore, in this application, the PCR primers may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to nucleic acids. Markers include, but are not limited to, dyes; radioactive markers, such as... 32 P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moieties (positive or negative) or, optionally, charge-neutral. The label can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the label is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).
[0016] Furthermore, in the aforementioned application, the PCR primers include a forward primer and a reverse primer, wherein the forward primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO: 2, and the reverse primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO: 3.
[0017] This application also provides PCR primers for identifying, assisting in the identification and / or comparing the cold resistance of wheat, the PCR primers consisting of a forward primer and a reverse primer, wherein the forward primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO: 2, and the reverse primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO: 3.
[0018] This application also provides kits for identifying, assisting in the identification and / or comparing the cold resistance of wheat, said kits containing any of the substances described above or said kits containing the PCR primers described above.
[0019] This application also provides the use of the kit in identifying, assisting in the identification and / or comparing cold resistance of wheat.
[0020] This application also provides a DNA molecule, the nucleotide sequence of which is SEQ ID NO: 1.
[0021] This application also provides a method for identifying, assisting in the identification, and / or comparing the cold resistance of wheat, the method comprising using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with the PCR primers or the kit to obtain PCR products, determining the genotype of the SNP molecular marker of the wheat to be tested based on the PCR products, and identifying, assisting in the identification, or comparing the cold resistance of wheat based on the genotype.
[0022] In the above method, the genotype can be GG or AA. GG is a homozygous type where the SNP molecular marker (deoxyribonucleotide at position 457 of SEQ ID NO: 1) is G, and AA is a homozygous type where the SNP molecular marker is A. The cold resistance of the tested wheat with genotype AA is higher or candidate higher than that of the tested wheat with genotype AA.
[0023] This application also provides a method for breeding cold-resistant wheat, comprising selecting a test wheat with the genotype AA of the SNP molecular marker as a parent and hybridizing it with another parent to obtain cold-resistant wheat, wherein the cold-resistant wheat has higher cold resistance than the other parent.
[0024] This application also provides applications of the SNP molecular marker, wherein the SNP molecular marker is used in any of the following:
[0025] Application of A1' in identifying or assisting in the identification of wheat cold resistance;
[0026] Application of A2') in the preparation of products for identifying or assisting in the identification of wheat cold resistance;
[0027] Application of A3') in comparing or assisting in comparing wheat cold resistance;
[0028] Application of A4') in the preparation of products for comparative or auxiliary comparison of wheat cold resistance;
[0029] The application of A5' in plant breeding;
[0030] The application of A6') in the preparation of products of the plant breeding;
[0031] Application of A7 as a molecular marker for cold resistance in wheat.
[0032] In this application, the product may be a reagent and / or a kit.
[0033] In this application, the indicator for plant breeding can be plant cold resistance.
[0034] In this application, the purpose of plant breeding may be to cultivate plants with improved cold resistance.
[0035] In this application, the purpose of plant breeding may also be to cultivate plants with reduced cold resistance.
[0036] The improved cold resistance can mean that the cold resistance is higher than that of the parent. The decreased cold resistance can mean that the cold resistance is lower than that of the parent.
[0037] In this application, the cold resistance may be antifreeze and / or cold resistance.
[0038] In this application, the cold-resistant wheat may be frost-resistant (freezing-resistant) wheat and / or cold-resistant (cold-resistant) wheat.
[0039] Plant cold resistance includes chilling resistance and freezing resistance. Damage to plants caused by temperatures above freezing is called chilling injury, and the plant's adaptation to temperatures above freezing is called chilling resistance. Damage to plants caused by temperatures below freezing is called freezing injury, and the plant's adaptation to temperatures below freezing is called freezing resistance.
[0040] In some embodiments of this application, cold resistance is divided into 5 cold resistance levels based on the survival rate of the wheat to be tested. Cold resistance level 1 means that 81%-100% of the wheat individuals in the material are frozen to death, cold resistance level 2 means that 61%-80% of the wheat individuals in the material are frozen to death, cold resistance level 3 means that 41%-60% of the wheat individuals in the material are frozen to death, cold resistance level 4 means that 21%-40% of the wheat individuals in the material are frozen to death, and cold resistance level 5 means that 0%-20% of the wheat individuals in the material are frozen to death.
[0041] In this application, the plant may be any of the following:
[0042] S1, Poaceae (grass family)
[0043] S2, plants of the genus *Wheat*,
[0044] S3, plants of the genus Aegilops.
[0045] S4, wheat,
[0046] S5, Goatgrass.
[0047] In this application, the wheat to be tested can be a wheat inbred line or a pure line.
[0048] The beneficial technical effects achieved by this application are as follows:
[0049] This application utilizes genome-wide association analysis to identify an SNP located at position 457 of the nucleotide sequence of SEQ ID NO.1 that is significantly associated with wheat cold resistance. The SNP molecular marker is not limited by the wheat growth stage and can be selected during the seedling stage, which greatly reduces the breeding workload, significantly shortens the wheat breeding selection cycle, and improves breeding efficiency. It can be used in practice for molecular marker-assisted breeding to improve wheat cold resistance. Attached Figure Description
[0050] Figure 1 Temperature records from the Beijing Changping test base for 2023-2024.
[0051] Figure 2 Manhattan plot of SNP markers that are highly associated with wheat cold resistance.
[0052] Figure 3 For the comparison of cold resistance differences among different genotypes of SNP markers that are highly associated with cold resistance in wheat in this application, the AA allele was significantly associated with strong cold resistance in wheat (P<0.001). Detailed Implementation
[0053] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.
[0054] 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 and reagents used in the following examples are commercially available.
[0055] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.
[0056] The following examples used GraphPad Prism statistical software to process the data. Experimental results are expressed as mean ± standard deviation. A t-test was used, with P < 0.05 (*) indicating a significant difference, and P < 0.05 indicating a statistically significant difference.
[0057] 0.001 (***) indicates a highly significant difference.
[0058] Example 1: Identifying SNPs Significantly Associated with Wheat Cold Resistance Based on Genome-wide Association Analysis (GWAS)
[0059] (1) The 309 wheat germplasm materials in the following implementation example were kindly provided by the He Fei research group of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and are disclosed in the literature "He F, Pasam R, Shi F, Kant S, Keeble-Gagnere G, Kay P, Forrest K, Fritz A, Hucl P, Wiebe K, Knox R, Cuthbert R, Pozniak C, Akhunova A, Morrell PL, Davies JP, Webb SR, Spangenberg G, Hayes B, Daetwyler H, Tibbits J, Hayden Mand Akhunov E (2019) Exome sequencing highlights the role of wild-relative introgression in shaping the adaptive landscape of the wheatgenome. Nature Genetics 51(5):896-904." The public can obtain the above biological materials from the applicant. The above biological materials obtained are only for repeating the experiments of this application and cannot be used for other purposes.
[0060] (2) 309 wheat germplasm resources were planted at the Pingxifu Farm of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (located in the Changping Experimental Base, Beijing) in 2023-2024. The experiment employed a split-plot design with three replicates. Each material was planted in three rows, with a row length of 1.5m and a row spacing of 0.25m, and 15 seeds per row. Sowing was carried out on September 25, 2023, and routine field management was implemented. No serious pests or diseases occurred during the growing season. Before the wheat turned green, the cold resistance of each germplasm material was investigated on March 15, 2024. The survival rate of each germplasm was used as the cold resistance index to evaluate cold resistance. Ten plants / rows were randomly selected from each germplasm, and the average value was taken. During the wheat overwintering period, field temperature records showed that from November 2023 to March 2024, there were 132 days with minimum temperatures below 0 degrees Celsius. Detailed temperature changes can be found in [link to relevant data]. Figure 1 .
[0061] Based on the survival rate of the wheat planting materials being tested, cold resistance was divided into 5 cold resistance levels. Cold resistance level 1 represents that 81%-100% of the wheat individuals in each germplasm were frozen to death; cold resistance level 2 represents that 61%-80% of the wheat individuals in each germplasm were frozen to death; cold resistance level 3 represents that 41%-60% of the wheat individuals in each germplasm were frozen to death; cold resistance level 4 represents that 21%-40% of the wheat individuals in each germplasm were frozen to death; and cold resistance level 5 represents that 0%-20% of the wheat individuals in each germplasm were frozen to death.
[0062] (3) Genomic DNA was extracted from 309 wheat samples. Genotyping was performed on these 309 wheat samples using exome sequencing. The minor allele loci of the genotypes were filtered (MAF<0.05). A total of 224,842 polymorphic SNP markers were detected after filtering.
[0063] (4) Genome-wide association analysis was performed using TASSEL 5.0 software. Kinship matrices were constructed and principal component analysis (PCA) was performed based on the filtered genotypes. The first three principal components were used for mixed linear model analysis. A Manhattan plot threshold of 6 was used as the criterion for selecting significant loci. The detected loci were significantly associated with wheat cold resistance.
[0064] (5) Figure 2 Manhattan plot of genome-wide association analysis (GWAS) using a mixed linear model to represent wheat cold resistance grades. The dashed line in the plot represents -Log10(p) = 4, and the dot indicated by the arrow represents the significant marker S5A_592434280 identified in this application. According to the Chinese Spring reference genome sequence information (Chinese Spring v2.1), this significant site S5A_592434280 is located at nucleotides 592,434,280 on chromosome 5AL. Compared with the GG genotype, wheat containing the AA genotype exhibits significantly enhanced cold resistance. Figure 3 ).
[0065] Example 2: Development of S5A_592434280 SNP tag
[0066] Based on the Chinese Spring reference genome sequence information (Chinese Spring v2.1), a total of 1000bp sequences were extracted from the upstream and downstream sequences of the SNP locus (S5A_592434280(G / A) or S5A_592434280SNP) that is significantly associated with wheat cold resistance. Primer sequences were designed using Primer 5 software, and PCR amplification was performed using primers S5A_592434280-F and S5A_592434280-R.
[0067] The nucleotide sequences of primers S5A_592434280-F and S5A_592434280-R are as follows:
[0068] S5A_592434280-F:5'-GGTACTTCCAGTAGTTTTATTCC-3' (SEQ ID NO: 2);
[0069] S5A_592434280-R: 5'-ATGCTTTTCAGTCGGAGTCAAC-3' (SEQ ID NO: 3).
[0070] The PCR amplification system is shown in Table 1:
[0071] Table 1. PCR amplification system
[0072] Total volume 10μL 50-100 ng / μL DNA 1.0μL forward primer 0.5μL reverse primer 0.5μL PCR Mix 5.0μL <![CDATA[ddH2O]]> 3.0μL
[0073] The PCR reaction procedure is shown in Table 2:
[0074] Table 2. PCR reaction procedure
[0075]
[0076] The amplified product was recovered and sequenced to obtain a nucleotide sequence containing the S5A_592434280 SNP site, as shown in SEQ ID No. 1. The SNP site is located at the 457th bp position from the 5' end of the sequence shown in SEQ ID NO: 1, and the base at this position is either G or A. The 'r' at the 457th bp position of SEQ ID NO: 1 represents G (guanine deoxyribonucleotide) or A (adenine deoxyribonucleotide).
[0077] Table 3. Nucleotide sequence of SEQ ID NO: 1
[0078]
[0079]
[0080] Example 3: Identification, auxiliary identification, or comparison of wheat cold resistance using S5A_592434280 SNP-specific PCR primers.
[0081] The genotypes of the S5A_592434280 SNP in the above 309 wheat germplasms were determined using the following method: Specific procedures are as follows:
[0082] (1) Extract DNA from the wheat sample to be tested;
[0083] (2) PCR amplification was performed using specific primer pairs (S5A_592434280-F and primer S5A_592434280-R) to obtain PCR products; the total amount of PCR products was 10 μL, including 1 μL DNA template, 5 μL PCR mix, 0.5 μL forward primer, 0.5 μL reverse primer, and 3 μL ddH2O. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 40 sec, for 35 cycles; 72℃ extension for 10 min.
[0084] (3) The PCR amplification products were detected by 1% agarose gel electrophoresis and then sequenced. The sequencing peak diagram was then examined to analyze the genotype of the wheat samples. When the PCR product showed a single peak at position 457bp (G), the sample was of the GG genotype; when the PCR product showed a single peak at position 457bp (A), the sample was of the AA genotype. The wheat samples with the AA genotype of S5A_592434280SNP showed higher or higher cold resistance than the wheat samples with the GG genotype of S5A_592434280SNP. Figure 3 ).
[0085] Figure 3 As shown in Table 4, the SNP molecular markers used in this application have a high accuracy rate in identifying wheat with strong cold resistance. The overall cold resistance of the tested wheat with the genotype AA of S5A_592434280SNP is higher than that of the tested wheat with the genotype GG of S5A_592434280SNP. That is, the A allele at the S5A_592434280SNP locus is highly significantly associated with strong cold resistance in wheat. The cold resistance of wheat with the genotype AA of S5A_592434280SNP is higher than, or potentially higher than, that of wheat with the genotype GG of S5A_592434280SNP. Therefore, wheat materials with strong cold resistance can be rapidly selected or predicted using the genotype of S5A_592434280SNP. Using wheat with the genotype AA from the S5A_592434280SNP to cross with another parent, the resulting hybrid wheat offspring showed higher or higher cold resistance than the other parent, which can accelerate the molecular design breeding process of wheat varieties.
[0086] Table 4. Different genotypes and cold resistance levels of wheat variety 309 in the S5A_592434280SNP.
[0087]
[0088]
[0089]
[0090]
[0091] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented 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 are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. Application of substances that detect SNP molecular markers in any of the following: A1) Application in identifying or assisting in the identification of wheat cold resistance; A2) Application in the preparation of products for identifying or assisting in the identification of wheat cold resistance; A3) Application in comparing or assisting in comparing the cold resistance of wheat; A4) Application in the preparation of products for comparing or assisting in comparing the cold resistance of wheat; A5) Applications in plant breeding; A6) Application in the preparation of plant breeding products; The SNP molecular marker is the 457th deoxyribonucleotide of SEQ ID NO: 1, which is G or A.
2. The application according to claim 1, characterized in that, The substance contains PCR primers for amplifying wheat genomic DNA fragments including the SNP molecular marker.
3. The application according to claim 2, characterized in that, The PCR primers include a forward primer and a reverse primer. The forward primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO: 2, and the reverse primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO:
3.
4. PCR primers for identifying, assisting in the identification of, and / or comparing wheat cold resistance, characterized in that: The PCR primers consist of a forward primer and a reverse primer. The forward primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO: 2, and the reverse primer is a single-stranded DNA with the nucleotide sequence SEQ ID NO:
3.
5. A kit for identifying, assisting in the identification of, and / or comparing the cold resistance of wheat, characterized in that: The kit contains the substance of any one of claims 1 to 3 or the kit contains the PCR primers of claim 4.
6. The use of the kit according to claim 5 in the identification, auxiliary identification and / or comparison of wheat cold resistance.
7. A DNA molecule, characterized by: The nucleotide sequence of the DNA molecule is SEQ ID NO:
1.
8. A method for identifying, assisting in the identification and / or comparing the cold resistance of wheat, comprising using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with the PCR primers of claim 4 or the kit of claim 5 to obtain PCR products, determining the genotype of the SNP molecular marker of the wheat to be tested in claim 1 based on the PCR products, and identifying, assisting in the identification or comparing the cold resistance of wheat based on the genotype.
9. A method for breeding cold-resistant wheat, comprising selecting a test wheat with the genotype AA of the SNP molecular marker described in claim 1 as a parent and hybridizing it with another parent to obtain cold-resistant wheat, wherein the cold-resistant wheat has higher cold resistance than the other parent.
10. The application of the SNP molecular marker as described in claim 1, wherein the application is the application of the SNP molecular marker in any of the following: Application of A1' in identifying or assisting in the identification of wheat cold resistance; Application of A2') in the preparation of products for identifying or assisting in the identification of wheat cold resistance; Application of A3') in comparing or assisting in comparing wheat cold resistance; Application of A4') in the preparation of products for comparative or auxiliary comparison of wheat cold resistance; Application of A5' in plant breeding; Application of A6' in the preparation of plant breeding products; Application of A7' as a molecular marker for cold resistance in wheat.