Development and application of a kasp marker associated with frost resistance in wheat

Abstract

The application discloses development and application of a KASP marker related to wheat frost resistance, relates to the technical field of biology, and relates to a method for screening or assisting in screening wheat frost-resistant germplasm, which comprises the following steps: detecting whether a wheat to be measured is genotype qFR-1B.1a or genotype qFR-1B.1b, and the frost resistance of the wheat of genotype qFR-1B.1a is greater than or greater than the frost resistance of the wheat of genotype qFR-1B.1b; the wheat of the genotype qFR-1B.1a is wheat with a GG homozygous genotype of a G101A SNP site; the wheat of the genotype qFR-1B.1b is wheat with an AA homozygous genotype of the G101A SNP site; and the G101A SNP site is the 101st nucleotide from the 5' end in a wheat genome, as shown in SEQ ID NO:1. The application has important theoretical significance and economic value for selecting wheat germplasm or breeding offspring materials with high frost resistance by using molecular marker assisted selection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly to development and application of a KASP marker related to wheat frost resistance. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world, and its overwintering frost resistance is directly related to the stability of yield. Traditional breeding methods select cold-resistant varieties by phenotypic screening, which has the limitations of long cycle, low efficiency and easy environmental interference. The development of molecular breeding technology provides a new way to accelerate the breeding of frost-resistant varieties, but the existing technology still faces multiple challenges: first, the wheat genome is large and complex, and frost resistance is regulated by multiple genes. Traditional molecular markers (such as SSR, RFLP) have insufficient polymorphism, making it difficult to accurately locate key sites; second, the QTL positioning method based on linkage disequilibrium is easily disturbed by genetic background, leading to separation of markers and target traits; in addition, conventional SNP genotyping technology (such as TaqMan probe method) is high in cost and low in throughput, making it difficult to meet the demand of large-scale germplasm resource screening. Therefore, the development of efficient and accurate molecular detection tools has become a key technical bottleneck in the field of wheat frost resistance breeding.

[0003] In recent years, the emergence of KASP (Competitive Allele-Specific PCR) technology has provided a revolutionary solution for wheat frost resistance site detection. This technology can perform high-throughput and low-cost double-allele genotyping of SNP sites through fluorescence competitive binding principle, and has the advantages of simple operation, intuitive results, and wide applicability. Compared with traditional molecular marker technology, KASP does not require the synthesis of specific fluorescent probes, and only needs to design universal primers to achieve simultaneous detection of multiple targets, significantly reducing detection costs. In wheat stress resistance research, KASP technology has been successfully applied to the development and application of drought resistance and salt tolerance related QTL (such as QDTRS.daas-4AL and Q-5A) functional markers. In addition, KASP technology is combined with BSA (Bulked Segregant Analysis), which quickly locates the stress tolerance gene interval by constructing extreme phenotype DNA pool and screening with high-density SNP chip, significantly shortening the breeding cycle. Therefore, the development of wheat frost resistance site-specific molecular markers based on KASP technology is of great significance for accelerating the breeding of frost-resistant varieties and ensuring food security. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a KASP marker related to wheat frost resistance.

[0005] To solve the above technical problems, the technical solutions adopted by the present application are as follows.

[0006] A method for screening or assisting in screening different frost resistance wheat, comprising the following steps: detecting whether the wheat to be tested is genotype qFR-1B.1a or genotype qFR-1B.1b, the frost resistance of the wheat of genotype qFR-1B.1a is stronger than or is a candidate for being stronger than the frost resistance of the wheat of genotype qFR-1B.1b;

[0007] The wheat of genotype qFR-1B.1a is wheat whose genotype at G101A SNP site is GG homozygote;

[0008] The wheat of genotype qFR-1B.1b is wheat whose genotype at G101A SNP site is AA homozygote;

[0009] The G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0010] Further preferably, the step of detecting whether the wheat to be tested is genotype qFR-1B.1a or genotype qFR-1B.1b is as follows:

[0011] (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;

[0012] 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;

[0013] (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-1B.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-1B.1b.

[0014] Further preferably, the step of detecting whether the wheat to be tested is genotype qFR-1B.1a or genotype qFR-1B.1b is as follows:

[0015] (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;

[0016] 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.

[0017] (b2) sequencing the PCR amplification product obtained in step (b1);

[0018] (b3) obtaining the genotype of the wheat to be tested according to the sequencing result obtained in step (b2).

[0019] A kit for identifying or assisting in identifying the frost resistance of wheat, comprising a substance for detecting whether the wheat to be tested is genotype qFR-1B.1a or genotype qFR-1B.1b;

[0020] The genotype qFR-1B.1a is a genotype of GG homozygote at the G101A SNP site;

[0021] The genotype qFR-1B.1b is a genotype of AA homozygote at the G101A SNP site;

[0022] The G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0023] Further preferably, the substance for detecting whether the wheat to be tested is genotype qFR-1B.1a or genotype qFR-1B.1b is a primer combination.

[0024] 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.

[0025] The molecular marker shown in SEQ ID NO: 1.

[0026] The above kit or the above molecular marker is used in identifying or assisting in identifying the frost resistance of wheat.

[0027] The above kit or the above molecular marker is used in screening or assisting in screening wheat with different frost resistance.

[0028] The above kit or the above molecular marker is used in wheat breeding.

[0029] The primer combination is used in directed breeding or assisting in directed breeding of wheat lines with high frost resistance, and 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.

[0030] The beneficial effects produced by the above technical solutions are that the application provides the KASP marker Kasp_qFR-1B.1 for identifying the allelic variations of qFR-1B.1a and qFR-1B.1b and the relationship between the KASP marker and the frost resistance of wheat, and the KASP marker in the application can be applied to molecular marker assisted selection of wheat frost resistance to quickly and efficiently screen wheat varieties (germplasm) with high frost resistance, thereby accelerating the breeding process of high-quality wheat new varieties. The KASP marker in the application can be applied to molecular marker assisted selection of wheat frost resistance to quickly screen wheat varieties (germplasm) with high frost resistance, thereby accelerating the breeding process of wheat frost resistance new varieties. The application has important theoretical significance and economic value for molecular marker assisted selection of wheat varieties with high frost resistance. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the KASP primer position of the two allelic types of the common wheat frost resistance related QTL qFR-1B.1 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 upstream and downstream primer positions of the KASP marker are marked with double underlines. The sequence in the figure is the sequence of SEQ ID NO: 1.

[0032] Figure 2 is a Kasp_FR-1B.1 marker detection result graph of a wheat variety / germplasm in the application.

[0033] Figure 3 is a schematic diagram of the correlation analysis results of the wheat germplasm with genotypes qFR-1B.1a and qFR-1B.1b and the average frost resistance of different wheat varieties or germplasms in different environments (‘*’ represents P<0.05, that is, the difference reaches a significant level; ‘****’ represents P<0.0001, 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; ZB_2023 represents sowing in Zibo City, Shandong Province in 2022-2023; and TS_2025 represents sowing in Tangshan City, Hebei Province in 2024-2025).

[0034] Figure 4 is a DNA extraction on-site photo collected by the application.

[0035] Figure 5 is a DNA quality detection-agarose gel preparation photo.

[0036] Figure 6 is a DNA concentration determination photo.

[0037] Figure 7 Figures 2023 to early spring 2025 in Shijiazhuang and Tangshan wheat field photos, A is the phenotype of different wheat varieties in Shijiazhuang in early spring 2023, B is the phenotype of different wheat varieties in Shijiazhuang in early spring 2024, C is the field investigation of different wheat varieties in Tangshan in early spring 2025. DETAILED DESCRIPTION

[0038] 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.

[0039] It should be understood that when used in the specification and the appended claims of the present application, the term "comprising" indicates the presence of the described 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.

[0040] It should also be understood that the term "and / or" as used in the specification and the appended claims of the present application means any combination of one or more of the associated listed items and all possible combinations, and includes these combinations.

[0041] In the present application, the reference to "one embodiment" or "some embodiments" and 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 specifically noted. The terms "comprising", "including", "having" and their variants, mean "including but not limited to", unless otherwise specifically noted.

[0042] 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.

[0043] The technical solutions of the present application will be described in detail below in combination with specific embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0044] Example 1, detecting different allelic types of QTL qFR-1B.1 related to winter resistance of wheat in overwintering period by KASP marker Kasp_FR-1B.1

[0045] The detection of different allelic types of QTL qFR-1B.1 related to winter resistance of wheat in overwintering period by KASP marker Kasp_FR-1B.1 is divided into two steps: PCR amplification and genotyping. The methods used in the examples 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 and Resources, Hebei Academy of Agriculture and Forestry Sciences.

[0046] (1) PCR amplification system and procedure. As shown in Figure 4 and Figure 5 , wheat leaf genomic DNA was extracted using TPS solution, and 100 ul ddH2O was added for dissolution. DNA quality detection was performed by 1% agarose gel electrophoresis, and the extracted DNA was required to have no obvious impurities, clear bands, and no degradation. After measuring the concentration of the DNA (as shown in Figure 6 ), the wheat genomic DNA was diluted to 28.3 ng / ul, and the diluted wheat genomic DNA was used as the template for PCR amplification.

[0047] KASP marker primer working solution preparation: KASP primers were designed according to the base SNP sequence of wheat frost resistance related QTL qFR-1B.1. The polymorphism of this SNP site is G / A base difference, and the primer sequence is shown in Table 1. 12 ul of each upstream primer (100 uM), 30 ul of downstream primer (100 uM) were respectively taken, and sterile ultrapure water was added to 100 ul as the primer working solution of KASP marker, which was stored at 4℃ for standby.

[0048] The PCR amplification system is: template DNA 2.5 ul, primer working solution 0.07 ul, 2x KASP MasterMix (LGC company, product number: KBS-2100-100-OLI) 2.5 ul.

[0049] The PCR reaction program is: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, annealing for 20 s (the first annealing temperature is 61℃, and the temperature decreases by 0.6℃ for each cycle) for a total of 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing for 1 min for a total of 26 cycles; 72℃ extension for 3 min, 4℃ storage.

[0050] (2) Genotyping. After the completion of the PCR reaction, the well plate was taken out and scanned for data 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 has FAM fluorescence, which is distributed near the x-axis; the A base type has HEX fluorescence, which is distributed near the y-axis; and samples with no detected signal are distributed near the origin (see Figure 2 ).

[0051] Table 1 Primer sequences of KASP marker Kasp_FR-1B.1 for detecting the frost resistance of wheat

[0052] Primer name Nucleotide sequence of the primers (5’-3’) and their position in the sequence listing Upstream primer Fl 5’ - gaaggtgaccaagttcatgctCACTAAGGCAGCACCGTCG-3’ (SEQ ID NO: 2) Upstream primer F2 5’ - gaaggtcggagtcaacggattCACTAAGGCAGCACCGTCA-3’ (SEQ ID NO: 3) Downstream primer R 5’ - TCGATTCACGGTCATTGGAGTAAT-3’ (SEQ ID NO: 4)

[0053] Example 2. Application of KASP marker Kasp_FR-1B.1 to detect the frost damage grade of common wheat varieties or germplasm during the overwintering period

[0054] Among the 196 wheat varieties or germplasms, 93 varieties / germplasms were of allelic type qFR-1B.1a, and 103 varieties / germplasms were of allelic type qFR-1B.1b. These 196 varieties / germplasms were sown in Shijiazhuang City, Hebei Province, in 2022-2023, 2023-2024, and 2024-2025, in Zibo City, Shandong Province, in 2022-2023, 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. Figure 7 ).

[0055] The frost damage grade of wheat during the overwintering period 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 the frost damage grade, grades 1, 1+, 2-, 2, 2+, 3-, 3, 3+, 4-, 4, and 4+ were replaced by integers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively. The larger the value, the more severe the frost damage to the wheat and the more sensitive the wheat to low temperatures, and the weaker the frost resistance. The mean frost damage grade of wheat in different years and different locations is shown in Table 2.

[0056] Table 2 KASP detection results of wheat frost resistance-related marker Kasp_FR-1B.1 and frost damage grade data

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Statistical results show that the average of the freezing injury grade of the wheat variety / germplasm carrying the allele type qFR-1B.1a is lower than that of the wheat variety / germplasm carrying the allele type qFR-1B.1b in different years and different places, and the difference between the two reaches a significant or extremely significant level (see Table 3). Figure 3

[0064] Table 3 Statistical analysis results of the relationship between the allele variation type of the QTL qFR-1B.1 of common wheat and the freezing injury grade

[0065]

[0066] Note: The statistical analysis adopts a two-tailed t-test; * represents that the difference reaches a significant level, and ** represents that the difference reaches an extremely significant level.

[0067] According to the SNP molecular site related to the freezing resistance of common wheat, the molecular site is located at the 101th 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-1B.1 for detecting the overwintering period freezing resistance of wheat is developed (Table 1, Figure 1 ). The type with FAM fluorescence distributed near the x axis is the wheat freezing resistance type (qFR-1B.1a), and the type with HEX fluorescence distributed near the y axis is the low-temperature sensitive type (qFR-1B.1b). Through the identification of the overwintering period phenotype of 196 common wheat varieties or germplasm resources in multiple years and multiple places, it is shown that the marker can accurately genotype the two allele types qFR-1B.1a and qFR-1B.1b. The molecular marker related to the overwintering period freezing resistance of wheat provided in the application can be used to detect whether the wheat variety or strain contains the freezing resistance site, and has a high value for the freezing resistance breeding of wheat. Meanwhile, the KASP molecular marker developed can greatly accelerate the breeding process of the wheat freezing resistance variety.

[0068] ​Although the embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, changes and alterations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0069] In the above embodiments, the description of each embodiment is focused on, and the part not described or recorded in a certain embodiment can be referred to the relevant description of other embodiments.

[0070] The above-described embodiments are only used to illustrate the technical solutions of the present application, rather than limit it; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing embodiments can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements 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 present application, and should be included in the protection scope of the present application.

Claims

1. A method for screening or assisting in screening wheat with different frost resistance, characterized in that, The steps include: determining whether the wheat to be tested is genotype qFR-1B.1a or genotype qFR-1B.1b, and whether the frost resistance of wheat with genotype qFR-1B.1a is stronger or candidate to be stronger than that of wheat with genotype qFR-1B.1b; The wheat with genotype qFR-1B.1a is a wheat with a genotype of GG homozygous at the G101A SNP locus; The wheat with genotype qFR-1B.1b is a homozygous AA wheat at the G101A SNP locus; The G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

2. The method according to claim 1, characterized in that, The steps for detecting whether the wheat to be tested is genotype qFR-1B.1a or qFR-1B.1b are as follows: (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; 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; (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-1B.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-1B.1b.

3. The method according to claim 1, characterized in that, The steps for detecting whether the wheat to be tested is genotype qFR-1B.1a or qFR-1B.1b are as follows: (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; 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; (b2) Take the PCR amplification product obtained in step (b1) and sequence it; (b3) Based on the sequencing results obtained in step (b2), obtain the genotype of the wheat to be tested.

4. A kit for identifying or assisting in the identification of wheat frost resistance, characterized in that, This includes substances used to detect whether the wheat being tested is genotype qFR-1B.1a or qFR-1B.1b; The genotype qFR-1B.1a at the G101A SNP site is GG homozygous; The genotype qFR-1B.1b at the G101A SNP locus is homozygous for AA. The G101A SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

5. The reagent kit according to claim 4, characterized in that, The substance used to detect whether the wheat genotype to be tested is qFR-1B.1a or qFR-1B.1b is a primer combination; 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.

6. The use of the kit according to any one of claims 4 to 5 in identifying or assisting in the identification of wheat frost resistance.

7. The use of the kit according to any one of claims 4 to 5 in screening or assisting in screening wheat with different frost resistance.

8. Application of primer combinations in the directional or assisted directional breeding of wheat varieties with high frost resistance, wherein 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.

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