KASP marker for auxiliary screening of cold-resistant wheat germplasm and application of KASP marker

Abstract

The invention discloses a KASP marker for auxiliary screening of cold-resistant wheat germplasm and application of the KASP marker, and relates to the technical field of biology, in particular to a method for screening or auxiliary screening of wheat cold-resistant germplasm, which comprises the following steps: detecting whether the genotype of wheat to be detected is genotype qFR-7A. 1a or genotype qFR-7A. 1b; the cold resistance of the wheat with the genotype qFR-7A. 1a is higher than that of the wheat with the genotype qFR-7A. 1b, or the cold resistance of the wheat with the genotype qFR-7A. 1a is higher than that of the wheat with the genotype The wheat of which the genotype is qFR-7A. 1a is wheat of which the genotype is GG homozygous based on a G101A SNP (Single Nucleotide Polymorphism) site; the wheat of which the genotype is qFR-7A. 1b is wheat of which the genotype is AA homozygous based on a G101A SNP (Single Nucleotide Polymorphism) site; the G101A SNP site is the 101 nucleotide from the 5'terminal of SEQ ID NO: 1 in a wheat genome. The molecular marker has important theoretical significance and economic value for molecular marker-assisted selection of wheat germplasm or breeding offspring materials with relatively high cold resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to KASP markers for assisting in screening cold-resistant wheat germplasm and application thereof. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world, and its yield and quality are directly affected by low temperature stress. Traditional cold-resistant germplasm screening mainly relies on field phenotype evaluation, which needs to go through multiple generations of natural low temperature environment verification, and has the problems of long cycle, high cost and easy disturbance by environmental fluctuations. For example, in winter wheat breeding, the cold resistance needs to be evaluated by overwintering survival rate, frost damage index and other indicators, but the phenotype data are easily affected by planting season, geographical location and management measures, resulting in low screening efficiency. In addition, cold resistance traits are usually co-regulated by multiple genes, and phenotype selection cannot accurately analyze the correlation between genotype and phenotype, which restricts the rapid development and utilization of cold-resistant germplasm resources. Therefore, it is an urgent need in the field of cold-resistant breeding to develop efficient and accurate molecular marker assisted screening technology.

[0003] As a new high-throughput genotyping platform, KASP (Competitive Allele-Specific PCR) technology has shown significant advantages in crop genetic improvement due to its high flexibility, low cost and high precision. This technology realizes the rapid detection of single nucleotide polymorphism by competitive binding of fluorescent probes to target SNP sites, and is suitable for genotype analysis of large-scale germplasm resources. Compared with traditional molecular markers (such as SSR or CAPS markers), KASP does not need to rely on specific restriction enzymes or probe design, has high standardization degree of operation process, and can be compatible with automated equipment, which significantly improves the genotyping efficiency. In the study of wheat cold resistance, KASP markers can accurately locate SNP sites closely linked to cold resistance genes, provide molecular basis for early generation selection, break through the bottleneck of phenotype screening, and accelerate the genetic improvement process of cold-resistant germplasm.

[0004] Previous studies have shown that wheat cold resistance is regulated by multiple QTL sites such as Fr-A1 and Fr-A2, but the reported molecular markers are mostly developed based on traditional breeding populations, which have the problems of insufficient polymorphism or poor cross-germplasm applicability. For example, some markers are stable in specific genetic backgrounds, but lack universality in global wheat germplasm resources, which limits their commercial application value. In addition, the cloning and functional verification of cold resistance genes still lag behind the breeding needs, resulting in insufficient development of precise markers based on gene function. KASP technology can construct a universal marker system across germplasm by targeting key SNP sites of known cold resistance related genes (such as CBF transcription factor family or cold response protein coding region), which can realize early and accurate screening of cold-resistant germplasm, and provide technical support for wheat stress breeding under the background of climate change. SUMMARY

[0005] The technical problem solved by the present application is to provide a KASP marker for assisting in screening cold-resistant wheat germplasm and application thereof.

[0006] To solve the above technical problems, the technical solutions adopted by the present application are as follows.

[0007] A method for screening or assisting in screening cold-resistant wheat germplasm, comprising the following steps: detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b, and the cold resistance of the wheat with genotype qFR-7A.1a is stronger than or is a candidate for being stronger than the cold resistance of the wheat with genotype qFR-7A.1b.

[0008] The wheat with genotype qFR-7A.1a is wheat with a GG homozygous genotype based on the G101A SNP site.

[0009] The wheat with genotype qFR-7A.1b is wheat with an AA homozygous genotype based on the G101A SNP site.

[0010] The G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0011] Further preferably, the step of detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b is as follows:

[0012] (a1) using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with a primer combination to obtain a PCR amplification product;

[0013] The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3, and a downstream primer R shown in SEQ ID NO: 4;

[0014] (a2) after step (a1) is completed, detecting the fluorescence signal of the PCR amplification product using a fluorescence quantitative PCR instrument, obtaining the genotype of the wheat to be tested according to the two fluorescence signal values and signal distribution, if the fluorescence signal distribution of the amplification product is close to the x-axis and far from the origin, i.e. consistent with the fluorescence of the fluorescent group labeled by primer F1, then the wheat sample to be tested is of genotype qFR-7A.1a; if the fluorescence signal distribution of the amplification product is close to the y-axis and far from the origin, i.e. consistent with the fluorescence of the fluorescent group labeled by primer F2, then the wheat sample to be tested is of genotype qFR-7A.1b.

[0015] Further preferably, the step of detecting whether the genotype of the wheat to be tested is the genotype qFR-7A.1a or the genotype qFR-7A.1b is as follows:

[0016] (b1) performing PCR amplification using a primer combination on the genomic DNA of the wheat to be tested to obtain a PCR amplification product;

[0017] The primer combination consists of an upstream primer F1 as shown in SEQ ID NO: 2, an upstream primer F2 as shown in SEQ ID NO: 3, and a downstream primer R as shown in SEQ ID NO: 4;

[0018] (b2) sequencing the PCR amplification product obtained in step (b1);

[0019] (b3) obtaining the genotype of the wheat to be tested according to the sequencing result obtained in step (b2).

[0020] A kit for identifying or assisting in identifying the cold resistance of wheat, comprising a substance for detecting whether the genotype of the wheat to be tested is the genotype qFR-7A.1a or the genotype qFR-7A.1b;

[0021] The genotype qFR-7A.1a is a genotype based on the G101A SNP site being GG homozygous;

[0022] The genotype qFR-7A.1b is a genotype based on the G101A SNP site being AA homozygous;

[0023] The G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0024] Further preferably, the substance for detecting whether the genotype of the wheat to be tested is the genotype qFR-7A.1a or the genotype qFR-7A.1b is a primer combination;

[0025] The primer combination consists of an upstream primer F1 as shown in SEQ ID NO: 2, an upstream primer F2 as shown in SEQ ID NO: 3, and a downstream primer R as shown in SEQ ID NO: 4.

[0026] The molecular marker shown in SEQ ID NO: 1.

[0027] The above kit or the above molecular marker is used for identifying or assisting in identifying the cold resistance of wheat.

[0028] The above kit or the above molecular marker is used for screening or assisting in screening wheat with different cold resistance.

[0029] The kit or the molecular marker is applied in wheat breeding.

[0030] The primer combination is applied in the directional breeding or assisted directional breeding of a wheat strain with high cold resistance, and the primer combination is composed of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3, and a downstream primer R shown in SEQ ID NO: 4.

[0031] The application has the beneficial effects that the KASP marker Kasp_FR-7A.1 for identifying the allelic variations of qFR-7A.1a and qFR-7A.1b and the relationship between the KASP marker and wheat cold resistance are provided, the KASP marker is applied to molecular marker assisted selection of wheat cold resistance, and a wheat variety (germplasm) with high cold resistance can be quickly and efficiently screened, so that the breeding process of a high-quality wheat new variety is accelerated. The molecular marker related to wheat cold resistance can realize efficient and accurate genotyping of the SNP site related to cold resistance, and provides a standardized and high-throughput molecular detection tool for wheat cold resistance breeding, and is expected to significantly improve the breeding efficiency and accuracy of cold-resistant varieties. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the KASP primer position of the two allelic types of the wheat cold resistance related QTL qFR-7A.1 of common wheat in the sequence shown in SEQ ID NO. 1 from the 101st base of the 5' end; the box represents the sequence shown in SEQ ID NO. 1 from the 101st base of the 5' end, and the positions of the upstream and downstream primers of the KASP marker are marked with double underlines. The sequence in the figure is the sequence of SEQ ID NO. 1.

[0033] Figure 2 is a Kasp_FR-7A.1 marker detection result graph of a wheat variety / strain of the application.

[0034] Figure 3 is a schematic diagram of the correlation analysis results of the wheat germplasm with genotypes qFR-7A.1a and qFR-7A.1b and the average cold resistance of different wheat varieties or germplasms in different environments (‘*’ represents P<0.05, that is, the difference reaches a significant level; ‘**’ represents P<0.01, that is, the difference reaches an extremely significant level; SJZ_2023, SJZ_2024 and SJZ_2025 represent sowing in Shijiazhuang City, Hebei Province in 2022-2023, 2023-2024 and 2024-2025, respectively; and TS_2025 represents sowing in Tangshan City, Hebei Province in 2024-2025).

[0035] Figure 4 is a field photo of collecting leaves to extract DNA.

[0036] Figure 5 A photograph of the agarose gel for DNA quality detection of the present application.

[0037] Figure 6 A photograph for DNA concentration determination of the present application.

[0038] Figure 7 A photograph of wheat field in early spring from 2023 to 2025 in Shijiazhuang and Tangshan, wherein A is a photograph of field phenotype of different wheat varieties in early spring in Shijiazhuang in 2023, B is a photograph of field phenotype of different wheat varieties in early spring in Shijiazhuang in 2024, and C is a photograph of field phenotype investigation of different wheat varieties in early spring in Tangshan in 2025. DETAILED DESCRIPTION

[0039] The following examples illustrate the present application in detail. The various raw materials and equipment used in the present application are all conventional commercially available products, which can be directly obtained by market purchase. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0040] It should be understood that the term "comprising" as used in the specification and the appended claims indicates the presence of the recited features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0041] It should also be understood that the term "and / or" as used in the specification and the appended claims indicates any combination of one or more of the associated listed items and all possible combinations, and includes these combinations.

[0042] In the present application, the reference to "one embodiment" or "some embodiments" or the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, the appearances of the phrases "in one embodiment", "in some embodiments", "in other embodiments", "in additional embodiments", and the like, in various places in the specification are not necessarily all referring to the same embodiment, unless otherwise noted. The terms "comprise", "comprising", "have", "having", "include", "including", and "contain", "containing", or variants thereof, mean "including but not limited to", unless otherwise noted.

[0043] In addition, in the description of the present application and the appended claims, the terms "first", "second", "third", and the like are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0044] The technical solutions of the present application will be described clearly and completely below in connection with specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the present application.

[0045] Example 1: Detection of different allelic types of QTL qFR-7A.1 related to winter survival cold resistance in wheat by KASP marker Kasp_FR-7A.1

[0046] The detection of different allelic types of QTL qFR-7A.1 related to winter survival cold resistance in wheat by KASP marker Kasp_FR-7A.1 is divided into two steps: PCR amplification and genotyping. The methods used in the example are conventional methods unless otherwise specified. Most of the wheat varieties / lines used are well-known varieties at home and abroad. The common wheat varieties / lines are preserved by the Wheat Research Center of the Institute of Crops, Soils and Agro-Environment, Hebei Academy of Agriculture and Forestry Sciences.

[0047] (1) PCR amplification system and procedure. As shown in Figure 4 and Figure 5 , the TPS solution is used to extract the genomic DNA of wheat leaves, 100ul ddH2O is added for dissolution, and 1% agarose gel electrophoresis is used for DNA quality detection. The extracted DNA requires no obvious impurities, clear bands, and no degradation. After measuring the concentration of the DNA (such as Figure 6 ), the wheat genomic DNA after dilution is used as a template for PCR amplification.

[0048] KASP marker primer working solution preparation: according to the base SNP sequence of the wheat cold resistance related QTL qFR-7A.1, the KASP primer is designed. The polymorphism of this SNP site is G / A base difference, and the primer sequence is shown in Table 1. 12ul of each upstream primer (100uM), 30ul of downstream primer (100uM) is respectively absorbed, and 100ul of sterile ultrapure water is supplemented to prepare the primer working solution of KASP marker, which is stored at 4℃ for standby use.

[0049] The PCR amplification system is as follows: 2.5ul of template DNA, 0.07ul of primer working solution, and 2.5ul of 2x KASP Master Mix (LGC Company, product number: KBS-2100-100-OLI).

[0050] PCR reaction program: 94°C pre-denaturation for 15 min; 94°C denaturation for 20 s, annealing for 20 s (the first annealing temperature is 61°C, and the temperature is reduced by 0.6°C for each cycle) for 10 cycles; 94°C denaturation for 20 s, 55°C annealing for 1 min for 26 cycles; 72°C extension for 3 min, 4°C storage.

[0051] (2) Genotyping. After the completion of the PCR reaction, the well plate was taken out and scanned using a fluorescence quantitative PCR instrument (ABI 7900). The fluorescence scanning results were graphically displayed using the R language "ggplot" package. The G base type had FAM fluorescence and was distributed near the x-axis; the A base type had HEX fluorescence and was distributed near the y-axis; samples with no detected signal were distributed near the origin (see FIG. 2). Figure 2 ).

[0052] Table 1 KASP markers for detecting cold resistance of wheat Kasp FR-7A.1 Primer sequence table

[0053]

[0054] Example 2. Application of KASP marker Kasp_FR-7A.1 to detect the freeze injury grade of common wheat varieties or germplasm during the overwintering period

[0055] Among the 227 wheat varieties or germplasms, 198 varieties / lines were of allelic type qFR-7A.1a, and 29 varieties / lines were of allelic type qFR-7A.1b. These 227 varieties / lines were sown in Shijiazhuang City, Hebei Province in 2022-2023, 2023-2024, and 2024-2025, and in Tangshan City, Hebei Province in 2024-2025, with a 2-meter row length, a randomized block design, and 2 replicates for each material (e.g., FIG. 1). Figure 7

[0056] ​The wintering freeze injury grade of wheat was evaluated according to the method described in the following reference: “Zhao Yong, Li Jiahao, Zhao Ruiling, Xu Ke, Xiao Yirao, Zhang Shuhua, Tian Jichun, Yang Xueju. Genome-wide association study reveals the genetic basis of cold tolerance in wheat, Molecular Breeding, 2020, 40:36”. For statistical analysis of freeze injury grade, 1, 1+, 2-, 2, 2+, 3-, 3, 3+, 4-, 4 and 4+ grades were replaced by integers 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 grades, respectively, and the larger the value represents the more serious the freeze injury of wheat and the more sensitive to low temperature and the weaker the cold resistance. The average freeze injury grade of wheat in different years and different locations is shown in Table 2.

[0057] Table 2 KASP detection results of wheat cold resistance related marker Kasp FR-7A.1 and freeze injury grade data

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] The statistical results show that the average freeze injury grade of wheat varieties / lines carrying allele type qFR-7A.1a in different years and different locations is lower than that of wheat varieties / lines carrying allele type qFR-7A.1b, and the difference between the two reaches a significant or extremely significant level (see Table 3). Figure 3

[0066] Table 3 Statistical analysis results of the relationship between QTL qFR-7A.1 allele variation type of common wheat and freeze injury grade

[0067] Note: The statistical analysis uses two-tailed t-test; * represents a significant difference, and ** represents an extremely significant difference. ​

[0068] The application is based on a cold resistance related SNP molecular site of common wheat, which is located at the 101st base from the 5' end of the sequence shown in SEQ ID NO. 1, and the polymorphism is G / A. According to the difference SNP site, a KASP marker Kasp_FR-7A.1 (Table 1, Figure 1 ) is developed for detecting the overwintering cold resistance of wheat. The type with FAM fluorescence distributed near the x axis is the cold resistance type (qFR-7A.1a), and the type with HEX fluorescence distributed near the y axis is the low temperature sensitive type (qFR-7A.1b). By identifying the overwintering phenotypes of 227 common wheat varieties or germplasm resources in multiple years and multiple sites, it is shown that the marker can accurately genotype the two allelic types of qFR-7A.1a and qFR-7A.1b. The molecular marker related to the cold resistance of wheat provided by the application can realize efficient and accurate genotyping of the cold resistance related SNP site, and provides a standardized and high-throughput molecular detection tool for wheat cold resistance breeding, which is expected to significantly improve the breeding efficiency and accuracy of cold resistance varieties.

[0069] Although the embodiments of the application have been shown and described, it should be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these examples without departing from the principles and spirit of the application, and the scope of the application is defined by the appended claims and their equivalents.

[0070] In the above embodiments, the description of each embodiment is focused on, and the parts not described or recorded in a certain embodiment can be referred to the related description of other embodiments.

[0071] The above described embodiments are only used to illustrate the technical solutions of the application, and not to limit it; although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitution for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application, and should be included in the protection scope of the application.

Claims

1. A method of screening or aiding in the screening of cold tolerant germplasm of wheat, characterized in that, The method comprises the following steps: detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b, and the cold resistance of the wheat with genotype qFR-7A.1a is stronger than or is a candidate for being stronger than the cold resistance of the wheat with genotype qFR-7A.1b; The wheat with genotype qFR-7A.1a is wheat with a GG homozygous genotype based on a G101A SNP site; The wheat with genotype qFR-7A.1b is wheat with an AA homozygous genotype based on a G101A SNP site; The G101A SNP site is the 101st nucleotide from the 5' end in SEQ ID NO: 1 in the wheat genome.

2. The method of claim 1, wherein, The step of detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b is as follows: (a1) using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with a primer combination to obtain a PCR amplification product; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3, and a downstream primer R shown in SEQ ID NO: 4; (a2) after step (a1) is completed, detecting the fluorescence signals of the PCR amplification product using a fluorescence quantitative PCR instrument, and obtaining the genotype of the wheat to be tested according to the two fluorescence signal values and signal distribution, if the fluorescence signal distribution of the amplification product is close to the x-axis and far from the origin, that is, consistent with the fluorescence of the fluorescent group labeled by primer F1, then the wheat sample to be tested is of genotype qFR-7A.1a; if the fluorescence signal distribution of the amplification product is close to the y-axis and far from the origin, that is, consistent with the fluorescence of the fluorescent group labeled by primer F2, then the wheat sample to be tested is of genotype qFR-7A.1b.

3. The method of claim 1, wherein, The step of detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b is as follows: (b1) using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with a primer combination to obtain a PCR amplification product; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3, and a downstream primer R shown in SEQ ID NO: 4; (b2) sequencing the PCR amplification product obtained in step (b1); (b3) obtaining the genotype of the wheat to be tested according to the sequencing result obtained in step (b2).

4. A kit for identifying or aiding in the identification of cold tolerance in wheat, characterized in that, The substance for detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b; The genotype qFR-7A.1a is a GG homozygous genotype based on a G101A SNP site; The genotype qFR-7A.1b is an AA homozygous genotype based on a G101A SNP site; The G101A SNP site is the 101st nucleotide from the 5' end in SEQ ID NO: 1 in the wheat genome.

5. The kit of claim 4, wherein The substance for detecting whether the genotype of the wheat to be tested is genotype qFR-7A.1a or genotype qFR-7A.1b is a primer combination; The primer combination consists of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3 and a downstream primer R shown in SEQ ID NO:

4.

6. The molecular marker shown in SEQ ID NO:

1.

7. Use of the kit of any one of claims 4 to 5 or the molecular marker of claim 6 in identifying or assisting in identifying cold resistance of wheat.

8. Use of the kit of any one of claims 4 to 5 or the molecular marker of claim 6 in screening or assisting in screening wheat with different cold resistance.

9. Use of the kit of any one of claims 4 to 5 or the molecular marker of claim 6 in wheat breeding.

10. Use of a primer combination consisting of an upstream primer F1 shown in SEQ ID NO: 2, an upstream primer F2 shown in SEQ ID NO: 3 and a downstream primer R shown in SEQ ID NO: 4 in targeted breeding or assisting in targeted breeding of wheat lines with high cold resistance.

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