KASP markers for assisting in the screening of cold-resistant wheat germplasm and their application

Abstract

This invention discloses KASP markers for assisting in the screening of cold-resistant wheat germplasm and their applications, relating to the field of biotechnology. A method for screening or assisting in the screening of cold-resistant wheat germplasm includes the following steps: detecting whether the genotype of the wheat to be tested is qFR-7A.1a or qFR-7A.1b; wheat with genotype qFR-7A.1a exhibits stronger or potentially stronger cold resistance than wheat with genotype qFR-7A.1b; the wheat with genotype qFR-7A.1a is a GG homozygous genotype based on the G101A SNP site; the wheat with genotype qFR-7A.1b is an AA homozygous genotype based on the G101A SNP site; the G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome. This invention has significant theoretical and economic value for using molecular markers to assist in the selection of wheat germplasm or breeding progeny materials with high cold resistance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the KASP marker for assisting in the screening of cold-resistant wheat germplasm and its application. Background Technology

[0002] Wheat (Triticum aestivum L.), as one of the world's most important food crops, is directly affected by low-temperature stress in terms of yield and quality. Traditional cold-resistant germplasm screening mainly relies on field phenotypic evaluation, which requires multiple generations of natural low-temperature environment verification, resulting in a long cycle, high cost, and susceptibility to environmental fluctuations. For example, in winter wheat breeding, cold resistance needs to be manually assessed through indicators such as overwintering survival rate and frost damage index, but phenotypic data are easily affected by planting season, geographical location, and management practices, leading to low screening efficiency. In addition, cold resistance traits are usually regulated by multiple genes in synergistic ways, and phenotypic selection makes it difficult to accurately analyze the relationship between genotype and phenotype, restricting the rapid development and utilization of cold-resistant germplasm resources. Therefore, developing efficient and accurate molecular marker-assisted screening technology has become an urgent need in the field of cold-resistant breeding.

[0003] Competitive allele-specific PCR (KASP) technology, as a novel high-throughput genotyping platform, has demonstrated significant advantages in crop genetic improvement due to its high flexibility, low cost, and high accuracy. This technology enables rapid detection of single nucleotide polymorphisms by competitively binding fluorescent probes to target SNP sites, making it suitable for genotyping analysis of large-scale germplasm resources. Compared to traditional molecular markers (such as SSR or CAPS markers), KASP does not rely on specific restriction endonucleases or probe design, has a high degree of standardization in its operation process, and is compatible with automated equipment, significantly improving genotyping efficiency. In wheat cold resistance research, KASP markers can accurately locate SNP sites closely linked to cold resistance genes, providing molecular evidence for early generation selection, overcoming the lag bottleneck of phenotypic screening, and accelerating the genetic improvement process of cold-resistant germplasm.

[0004] Existing research indicates that wheat cold resistance is regulated by multiple QTL sites, such as Fr-A1 and Fr-A2. However, most reported molecular markers are developed based on traditional breeding populations, resulting in insufficient polymorphism or poor cross-germplasm applicability. For example, some markers are stable in specific genetic backgrounds but lack universality across global wheat germplasm resources, limiting their commercial application value. Furthermore, the cloning and functional validation of cold resistance genes lag behind breeding needs, leading to insufficient development of precise gene function-based markers. KASP technology, by targeting key SNP sites (such as the CBF transcription factor family or cold-response protein coding regions) of known cold resistance-related genes, can construct a universal marker system across germplasm, enabling early and precise screening of cold-resistant germplasm and providing technical support for wheat stress-resistance breeding in response to climate change. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a KASP marker for assisting in the screening of cold-resistant wheat germplasm and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

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

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

[0009] The wheat with genotype qFR-7A.1b is a homozygous AA 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] More preferably, the step of detecting whether the genotype of the wheat to be tested is qFR-7A.1a or qFR-7A.1b is as follows:

[0012] (a1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using primer combinations to obtain PCR amplification products;

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

[0014] (a2) After completing step (a1), the fluorescence signal of the PCR amplification product is detected using a real-time PCR instrument. The genotype of the wheat to be tested is obtained based on 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 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 genotype qFR-7A.1b.

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

[0016] (b1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using primer combinations to obtain PCR amplification products;

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

[0018] (b2) Take the PCR amplification product obtained in step (b1) and sequence it;

[0019] (b3) Based on the sequencing results obtained in step (b2), obtain the genotype of the wheat to be tested.

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

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

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

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

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

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

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

[0027] Application of the above-mentioned kits or molecular markers in identifying or assisting in the identification of wheat cold resistance.

[0028] Application of the above-mentioned kits or molecular markers in screening or assisting screening of different cold-resistant wheat varieties.

[0029] Application of the above-mentioned kits or molecular markers in wheat breeding.

[0030] The primer combination is used in the targeted breeding or assisted targeted breeding of wheat varieties with high cold resistance. The primer combination consists of upstream primer F1 shown in SEQ ID NO: 2, upstream primer F2 shown in SEQ ID NO: 3, and downstream primer R shown in SEQ ID NO: 4.

[0031] The beneficial effects of adopting the above technical solution are as follows: This invention provides the KASP marker Kasp_FR-7A.1 for identifying qFR-7A.1a and qFR-7A.1b allelic variations and its correlation with wheat cold resistance. Applying the KASP marker of this invention to marker-assisted selection for wheat cold resistance can rapidly and efficiently screen wheat varieties (germplasm) with higher cold resistance, thereby accelerating the breeding process of high-quality new wheat varieties. The molecular markers related to wheat cold resistance provided by this invention can achieve efficient and accurate genotyping of cold resistance-related SNP sites, providing a standardized, 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. Attached Figure Description

[0032] Figure 1 This invention shows the KASP primer positions for the two allelic types of the common wheat cold-resistance-related QTL qFR-7A.1 at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1; the boxes represent the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, and the upstream and downstream primer positions of the KASP marker are indicated by double underscores. The sequence in the figure is the sequence of SEQ ID NO.1.

[0033] Figure 2 This is a diagram showing the marker detection results for the wheat variety / strain Kasp_FR-7A.1 of this invention.

[0034] Figure 3 This diagram illustrates the association between the mean cold resistance of wheat germplasm with genotypes qFR-7A.1a and qFR-7A.1b in different wheat varieties or germplasms under different environments ("*" indicates P<0.05, i.e., the difference reached a significant level; "**" indicates P<0.01, i.e., the difference reached a highly significant level; SJZ_2023, SJZ_2024, and SJZ_2025 represent wheat sown in Shijiazhuang City, Hebei Province in 2022-2023, 2023-2024, and 2024-2025, respectively; TS_2025 represents wheat sown in Tangshan City, Hebei Province in 2024-2025).

[0035] Figure 4 These are photos taken at the scene of DNA extraction from leaves for this invention.

[0036] Figure 5 Photograph of the DNA quality detection-agarose gel preparation for this invention.

[0037] Figure 6 This is a photograph of the DNA concentration measurement of this invention.

[0038] Figure 7 These are field photos of wheat in Shijiazhuang and Tangshan from early spring 2023 to early spring 2025. Among them, A is a field phenotypic photo of different wheat varieties in Shijiazhuang in early spring 2023, B is a field phenotypic photo of different wheat varieties in Shijiazhuang in early spring 2024, and C is a field phenotypic survey photo of different wheat varieties in Tangshan in early spring 2025. Detailed Implementation

[0039] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0040] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0041] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0042] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0043] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0044] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1: Detection of different allelic types of the QTL qFR-7A.1 related to wheat winter hardiness using the KASP marker Kasp_FR-7A.1.

[0046] The detection of different allelic types of the QTL qFR-7A.1, which regulates winter hardiness resistance in wheat, using the KASP marker Kasp_FR-7A.1 involved two steps: PCR amplification and genotyping. Unless otherwise specified, all methods used in the examples are conventional. Most of the wheat varieties / lines used were well-known domestic and international varieties. Common wheat varieties / lines were preserved by the Wheat Research Center of the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences.

[0047] (1) PCR amplification system and procedure. For example... Figure 4 and Figure 5 As shown, genomic DNA was extracted from wheat leaves using TPS solution, dissolved in 100 μL of ddH2O, and the DNA quality was determined by 1% agarose gel electrophoresis. The extracted DNA was required to be free of obvious impurities, have clear bands, and show no degradation. DNA concentration was then measured (e.g., ...). Figure 6 The sample was uniformly diluted to 28.3 ng / μl, and PCR amplification was performed using the diluted wheat genomic DNA as a template.

[0048] Preparation of KASP-labeled primer working solution: KASP primers were designed based on the SNP sequence of wheat cold resistance-related QTL qFR-7A.1, which exhibits a G / A base difference. The primer sequences are shown in Table 1. 12 μl each of the two upstream primers (100 μM) and 30 μl of the downstream primer (100 μM) were added to a final volume of 100 μl with sterile ultrapure water to prepare the KASP-labeled primer working solution, which was stored at 4°C for later use.

[0049] The PCR amplification system consisted of: 2.5 μl template DNA, 0.07 μl primer working solution, and 2.5 μl 2×KASP Master Mix (LGC, product number: KBS-2100-100-OLI).

[0050] The PCR reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s and annealing for 20 s (the first annealing temperature was 61℃, and the temperature was reduced by 0.6℃ for each cycle) for a total of 10 cycles; 94℃ denaturation for 20 s and 55℃ annealing for 1 min for a total of 26 cycles; 72℃ extension for 3 min and storage at 4℃.

[0051] (2) Genotyping. After the PCR reaction was completed, the well plates were removed, and data were scanned using a quantitative real-time PCR instrument (ABI 7900). The fluorescence scan results were graphically displayed using the R language "ggplot" package. G bases showed FAM fluorescence, distributed near the x-axis; A bases showed HEX fluorescence, distributed near the y-axis; samples with no detected signal were distributed near the origin (see attached diagram). Figure 2 ).

[0052] Table 1. KASP markers used to detect cold resistance in wheat. Kasp_FR-7A.1 Primer sequence listing

[0053]

[0054] Example 2: Application of the KASP marker Kasp_FR-7A.1 to detect the overwintering frost damage level of common wheat varieties or germplasm.

[0055] Of the 227 wheat varieties or germplasm accessions, 198 were of allele type qFR-7A.1a, and 29 were of allele 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 2-meter row lengths. A randomized block design was used, with two replicates for each material (e.g., ...). Figure 7 ).

[0056] The wintering frost damage severity of wheat was assessed 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 the statistical analysis of frost damage severity, levels 1, 1+, 2-, 2, 2+, 3-, 3, 3+, 4-, 4, and 4+ were replaced with integers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively. Higher values ​​indicate more severe frost damage, greater sensitivity to low temperatures, and weaker cold resistance. The average frost damage severity of wheat in different years and locations is shown in Table 2.

[0057] Table 2. Results of KASP test for wheat cold resistance-related markers and data on frost damage levels.

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Statistical results show that wheat varieties / lines carrying allelic type qFR-7A.1a had lower mean frost damage severity in different years and locations than wheat varieties / lines carrying allelic type qFR-7A.1b, with significant or highly significant differences between the two groups (see attached). Figure 3 (Table 3).

[0066] Table 3. Statistical analysis results of the relationship between QTLqFR-7A.1 allele variation type and frost damage severity in common wheat.

[0067] Note: Statistical analysis uses a two-tailed t-test; * indicates that the difference is significant, and ** indicates that the difference is highly significant.

[0068] This invention utilizes a SNP molecular site related to cold resistance in common wheat, specifically the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, with a polymorphism of G / A. Based on this differential SNP site, a KASP marker, Kasp_FR-7A.1, was developed for detecting cold resistance in wheat during winter (Table 1). Figure 1 The allelic markers carrying FAM fluorescence and distributed near the x-axis indicate the cold-resistant type (qFR-7A.1a), while those carrying HEX fluorescence and distributed near the y-axis indicate the low-temperature sensitive type (qFR-7A.1b). Analysis of the overwintering phenotypes of 227 common wheat varieties or germplasm resources over multiple years and at multiple locations showed that this marker can accurately classify the two allelic types, qFR-7A.1a and qFR-7A.1b. The molecular markers related to wheat cold resistance provided by this invention enable efficient and accurate classification of cold-resistant SNP loci, providing a standardized, high-throughput molecular detection tool for wheat cold resistance breeding, and is expected to significantly improve the efficiency and accuracy of cold-resistant variety breeding.

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

[0070] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0071] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

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 of genotype qFR-7A.1a is stronger than or is a candidate for being stronger than the cold resistance of the wheat of genotype qFR-7A.1b; The wheat of genotype qFR-7A.1a is wheat with a GG homozygous genotype based on a G101A SNP site; The wheat of 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. 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 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. 5.A molecular marker related to cold resistance of wheat, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the 101st nucleotide from the 5' end of SEQ ID NO: 1 is a SNP site, and the polymorphism is G / A. 6.Use of the kit of claim 4 or the molecular marker of claim 5 in identifying or assisting in identifying the cold resistance of wheat. 7.Use of the kit of claim 4 or the molecular marker of claim 5 in screening or assisting in screening wheat with different cold resistance. 8.Use of the kit of claim 4 or the molecular marker of claim 5 in wheat breeding. 9.Use of a primer combination in targeted breeding or assisting in targeted breeding of wheat lines with high cold resistance, the primer combination consisting 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.

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