SNP marker on short arm of wheat chromosome 2a associated with cold resistance and application thereof

Abstract

The application discloses a SNP marker related to cold resistance on the short arm of wheat 2A chromosome and application thereof, and belongs to the field of biotechnology and molecular breeding. In order to identify, assist in identifying and / or compare the cold resistance of wheat, the application provides application of a SNP molecular marker, wherein the SNP molecular marker is a deoxyribonucleotide at position 361 of SEQ ID NO:1, which is G or A, and the application can be application of the SNP molecular marker in identifying or assisting in identifying the cold resistance of wheat. By using whole genome association analysis, the application identifies that a SNP at position 361 of the nucleotide sequence of SEQ ID NO.1 is significantly related to the cold resistance of wheat, the SNP molecular marker is not limited by the growth stage of wheat, and selection can be performed at the seedling stage, so that the selection period of wheat breeding is significantly shortened, the breeding efficiency is improved, and the SNP molecular marker assisted breeding for improving the cold resistance of wheat can be used in practice.

Description

Technical Field

[0001] This application relates to the fields of biotechnology and molecular breeding, specifically to a cold-resistance-related SNP marker on the short arm of wheat chromosome 2A and its application. Background Technology

[0002] Wheat is one of the world's most important food crops and my country's second largest food crop. Its high and stable yields directly affect people's living standards and national food security. In recent years, frequent low temperatures and cold waves have posed a serious threat to wheat production, becoming one of the main disasters causing wheat yield losses. Therefore, improving wheat's cold resistance has always been an important breeding goal.

[0003] Molecular marker-assisted selection breeding technology utilizes molecular markers that control the linkage of target trait loci / genes, and achieves the purpose of selecting target traits through molecular biological detection of genotypes. Its advantages are simplicity and speed, and it is not affected by the environment and its interactions, enabling rapid and efficient breeding of target resource materials.

[0004] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide in the genome, including transitions, transversions, deletions, and insertions. SNP markers have the following main characteristics: 1. Numerous and widely distributed; 2. Suitable for rapid, large-scale screening; 3. Easy estimation of allele frequencies; 4. Easy genotyping. Summary of the Invention

[0005] The technical problem this application aims to solve is: how to identify, assist in identifying, and / or compare the cold resistance of wheat. To solve the above technical problem, this application provides the following technical solution:

[0006] This application provides the application of SNP molecular markers, wherein the SNP molecular marker may be the 361st deoxyribonucleotide of SEQ ID NO: 1, which is G (guanine deoxyribonucleotide) or A (adenine deoxyribonucleotide), and the application may be the application of the SNP molecular marker in any of the following:

[0007] A1) Application in identifying or assisting in the identification of wheat cold resistance;

[0008] A2) Application in the preparation of products for identifying or assisting in the identification of wheat cold resistance;

[0009] A3) Application in comparing or assisting in comparing the cold resistance of wheat;

[0010] A4) Application in the preparation of products for comparing or assisting in comparing the cold resistance of wheat;

[0011] A5) Applications in plant breeding;

[0012] A6) Application in the preparation of plant breeding products;

[0013] Application of A7 as a molecular marker for cold resistance in wheat.

[0014] In this application, the indicator for plant breeding can be plant cold resistance.

[0015] In this application, the purpose of plant breeding may be to cultivate plants with improved cold resistance.

[0016] In this application, the purpose of plant breeding may also be to cultivate plants with reduced cold resistance.

[0017] The improved cold resistance can mean that the cold resistance is higher than that of the parent.

[0018] The reduced cold resistance may mean that the cold resistance is lower than that of the parent.

[0019] In this application, the plant may be any of the following:

[0020] S1, Poaceae (grass family)

[0021] S2, plants of the genus *Wheat*,

[0022] S3, plants of the genus Aegilops.

[0023] S4, wheat,

[0024] S5, Goatgrass.

[0025] This application also provides the use of substances for detecting the SNP molecular markers in any of the following:

[0026] Application of A1' in identifying or assisting in the identification of wheat cold resistance;

[0027] Application of A2') in the preparation of products for identifying or assisting in the identification of wheat cold resistance;

[0028] Application of A3') in comparing or assisting in comparing wheat cold resistance;

[0029] Application of A4') in the preparation of products for comparative or auxiliary comparison of wheat cold resistance;

[0030] The application of A5' in plant breeding;

[0031] The application of A6' in the preparation of products of the plant breeding.

[0032] In this application, the product may be a reagent and / or a kit.

[0033] 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.

[0034] Furthermore, the substance contains PCR primers for amplifying wheat genomic DNA fragments including the SNP molecular marker.

[0035] 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).

[0036] Furthermore, the PCR primers include a forward primer and a reverse primer, wherein the forward primer may be a single-stranded DNA with the nucleotide sequence SEQ ID NO: 2, and the reverse primer may be a single-stranded DNA with the nucleotide sequence SEQ ID NO: 3.

[0037] This application also provides PCR primers for identifying, assisting in the identification and / or comparing the cold resistance of wheat, wherein the PCR primers consist of a forward primer and a reverse primer, wherein the forward primer may be a single-stranded DNA with the nucleotide sequence SEQ ID NO: 2, and the reverse primer may be a single-stranded DNA with the nucleotide sequence SEQ ID NO: 3.

[0038] This application also provides kits for identifying, assisting in the identification and / or comparing wheat cold resistance, said kits containing the substances described above and / or said kits containing the PCR primers described above.

[0039] This application also provides the use of the kit in identifying, assisting in the identification and / or comparing cold resistance of wheat.

[0040] This application also provides a DNA molecule, the nucleotide sequence of which is SEQ ID NO: 1.

[0041] This application also provides a method for identifying, assisting in the identification, or comparing the cold resistance of wheat. The method includes using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with the above-mentioned PCR primers or the above-mentioned kit to obtain PCR products, determining the genotype of the above-mentioned 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.

[0042] In the method, the genotype can be GG or AA. GG is a homozygous genotype where the SNP molecular marker (deoxyribonucleotide at position 361 of SEQ ID NO: 1) is G (guanine deoxyribonucleotide), and AA is a homozygous genotype where the SNP molecular marker is A (adenine deoxyribonucleotide). The cold resistance of the tested wheat with genotype GG is higher or potentially higher than that of the tested wheat with genotype AA.

[0043] This application also provides a method for breeding cold-resistant wheat, comprising selecting a test wheat with the genotype GG 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.

[0044] Furthermore, in this application, the cold resistance may be antifreeze and / or cold resistance.

[0045] Furthermore, in this application, the cold-resistant wheat may be frost-resistant (freezing-tolerant) wheat and / or cold-resistant (cold-tolerant) wheat.

[0046] 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.

[0047] 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.

[0048] In this application, the wheat to be tested can be a wheat inbred line or a pure line.

[0049] The beneficial technical effects achieved by this application are as follows:

[0050] This application utilizes genome-wide association analysis to identify an SNP located at position 361 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

[0051] Figure 1 Temperature records from the Beijing Changping test base for 2023-2024.

[0052] Figure 2 Manhattan plot of SNP markers that are highly associated with wheat cold resistance.

[0053] 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 GG allele was significantly associated with strong cold resistance in wheat (P = 4.7e-14). Detailed Implementation

[0054] This application selects cold-resistant wheat materials by choosing favorable allelic variations of SNP markers, which can significantly improve selection efficiency and shorten the breeding period of cold-resistant wheat varieties, and is of great significance to increasing wheat yield.

[0055] 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.

[0056] 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.

[0057] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0058] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The t-test is used, and P < 0.05 indicates a significant difference, and P < 0.001 indicates a highly significant difference.

[0059] Example 1: Identification, auxiliary identification, or comparison of wheat cold resistance using SNP molecular markers.

[0060] 1. Based on genome-wide association analysis (GWAS), identify SNP loci significantly associated with wheat cold resistance.

[0061] (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.

[0062] (2) 309 wheat germplasm materials were sourced from wheat-producing regions in different countries worldwide. They 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. A split-plot design with three replicates was used. Each germplasm material was planted in three rows, 1.5m long, with a row spacing of 0.25m, and 15 seeds per row. Sowing was on September 25, 2023, and conventional field management was implemented. No serious pests or diseases occurred during the growing season. Before the wheat turned green, the survival rate of each germplasm material was investigated on March 15, 2024. The survival rate of each germplasm was used as a cold resistance index to evaluate cold resistance. Ten plants / rows were randomly selected from each germplasm material in each replicate, 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 .

[0063] 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.

[0064] (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.

[0065] (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.

[0066] (5) Figure 2Manhattan 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) = 6, and the dot indicated by the arrow represents the significant marker S2A_5489336 identified in this application. According to the Chinese Spring v2.1 reference genome sequence, this significant site S2A_5489336 is located at nucleotide 5,489,336 on chromosome 2AS. Compared to the AA genotype, wheat containing the GG genotype exhibits significantly enhanced cold resistance. Figure 3 ).

[0067] 2. S2A_5489336SNP tag development

[0068] Based on the Chinese Spring reference genome sequence information (Chinese Spring v2.1), a total of 800 bp sequences were extracted from the upstream and downstream sequences of the SNP site (S2A_5489336(G / A)) that is significantly associated with wheat cold resistance. Primers specific to the SNP (S2A_5489336(G / A)) were designed using Primer 5 software, including forward and reverse primers. The nucleotide sequences are as follows:

[0069] S2A_5489336-F (forward primer): 5'-AGCAGCTGATCATCTTATTGCAT-3' (SEQ IN NO: 2);

[0070] S2A_5489336-R (reverse primer): 5'-CTGCTGCTGCTGCCACTGCG-3' (SEQ IN NO: 3);

[0071] Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using forward and reverse primers as amplification primer pairs.

[0072] The PCR amplification system is shown in Table 1:

[0073] Table 1. PCR amplification system

[0074] Total volume 10μL 100 ng / μL DNA 1.0μL forward primer 0.5μL reverse primer 0.5μL PCR Mix 5.0μL <![CDATA[ddH2O]]> 3.0μL

[0075] The PCR reaction procedure is shown in Table 2:

[0076] Table 2. PCR reaction procedure

[0077]

[0078] The amplified product was recovered and sequenced to obtain the nucleotide sequence containing the aforementioned SNP (S2A_5489336(G / A)), as shown in SEQ ID NO: 1. S2A_5489336(G / A) is located at the 361 bp position from the 5' end of the sequence shown in SEQ ID NO: 1, and the deoxynucleotide at this position is either G (guanine deoxyribonucleotide) or A (adenine deoxyribonucleotide). The 'r' at position 361 of SEQ ID NO: 1 represents guanine deoxyribonucleotide (G) or adenine deoxyribonucleotide (A).

[0079] Table 3. Nucleotide sequence of SEQ ID NO: 1

[0080]

[0081] 3. Use SNP (S2A_5489336(G / A)) specific PCR primers to identify, assist in identifying, or compare wheat cold resistance.

[0082] The genotype of S2A_5489336(G / A) in the above 309 wheat germplasms was determined using the following method: Specific procedures are as follows:

[0083] (1) Extract DNA from wheat leaves to be tested;

[0084] (2) PCR amplification was performed using the PCR primer pair (S2A_5489336-F and primer S2A_5489336-R) to obtain the PCR product; the product consisted of 1 μL DNA template, 5 μL PCR mix, 0.5 μL forward primer, 0.5 μL reverse primer, and 3 μL ddH2O, for a total of 10 μL. 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.

[0085] (3) The PCR amplification products were detected by 1% agarose gel electrophoresis and then sequenced. After that, the sequencing peak diagram was viewed and the genotype of the wheat samples to be tested was analyzed. The genotype of wheat homozygous for S2A_5489336(G / A) (the 361st deoxyribonucleic acid of SEQ ID NO: 1) is GG, and the genotype of wheat homozygous for S2A_5489336(G / A) (the 361st deoxyribonucleic acid of SEQ ID NO: 1) is AA.

[0086] The results showed that among the 309 wheat germplasms, 278 germplasms had the genotype AA at the S2A_5489336(G / A) locus, and 31 germplasms had the genotype GG at the S2A_5489336(G / A) locus. The genotypic and phenotypic data of the 309 germplasms are shown in Table 4. The results indicate that the cold resistance of the wheat samples with the genotype GG at the S2A_5489336(G / A) locus was higher or potentially higher than that of the wheat samples with the genotype AA at the S2A_5489336(G / A) locus. In other words, compared to the wheat samples with the genotype AA at the S2A_5489336(G / A) locus, the wheat samples with the genotype GG at the S2A_5489336(G / A) locus exhibited higher cold resistance.

[0087] Figure 3 The results in Table 4 indicate that 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 GG genotype at the S2A_5489336(G / A) locus is higher than that of the tested wheat with the AA genotype. This means that the genotype at the S2A_5489336(G / A) locus is highly significantly correlated with strong cold resistance in wheat. By selecting the S2A_5489336(G / A) locus as the GG genotype, wheat materials with strong cold resistance can be quickly selected or predicted. Crossbreeding wheat parents with the S2A_5489336(G / A) locus as the GG genotype with another parent resulted in hybrid offspring with cold resistance higher than or potentially higher than that of the other parent, accelerating the molecular design breeding process for wheat varieties.

[0088] Table 4. Different genotypes and cold resistance grades of wheat variety 309 in the S2A_5489336SNP.

[0089]

[0090]

[0091]

[0092]

[0093] 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. The application of SNP molecular markers, characterized by: The SNP molecular marker is the 361st deoxyribonucleotide of SEQ ID NO: 1, which is G or A, and the application is the application of the SNP molecular marker 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; Application of A7 as a molecular marker for cold resistance in wheat.

2. To detect the use of the SNP molecularly labeled substance as described in claim 1 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.

3. 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.

4. The application according to claim 3, 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.

5. 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.

6. A kit for identifying, assisting in the identification, and / or comparing the cold resistance of wheat, characterized in that: The kit contains the substance described in any one of claims 2 to 4 and / or the kit contains the PCR primers described in claim 5.

7. The use of the kit according to claim 6 in the identification, auxiliary identification and / or comparison of wheat cold resistance.

8. A DNA molecule, characterized by: The nucleotide sequence of the DNA molecule is SEQ ID NO:

1.

9. 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 5 or the kit of claim 6 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.

10. A method for breeding cold-resistant wheat, comprising selecting a wheat with the genotype GG 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.

Images