Molecular marker for auxiliary screening of anti-freezing wheat germplasm and application thereof

Abstract

The invention discloses a molecular marker for auxiliary screening of anti-freezing wheat germplasm and application of the molecular marker, and relates to the technical field of biology, in particular to a method for screening or auxiliary screening of wheat with different freezing resistance, which comprises the following steps: detecting whether the genotype of wheat to be detected is qFDG-7A. 3a or qFDG-7A. 3b, and the freezing resistance gt of the wheat with the genotype qFDG-7A. 3a; the freezing resistance of the wheat with the genotype qFDG-7A. 3b; the wheat of which the genotype is qFDG-7A. 3a is wheat of which the genotype is AA homozygous based on an A129C SNP (Single Nucleotide Polymorphism) site; the wheat of which the genotype is qFDG-7A. 3b is wheat of which the genotype is CC homozygous based on an A129C SNP (Single Nucleotide Polymorphism) site; the A129C SNP site is the 129th nucleotide from the 5'tail end 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 progeny materials with high anti-freezing capacity.

Description

[0001] The present invention relates to the field of biotechnology, in particular to molecular markers for assisting in screening antifreeze wheat germplasm and their applications. Background Art

[0002] Wheat (Triticum aestivum L.), as one of the three major food crops in the world, its stable production is crucial for food security. However, low-temperature freezing injury has become an important environmental factor threatening wheat yield and quality. According to statistics, the reduction in yield caused by freezing injury in the main winter wheat producing areas of China can reach 5%-20% annually, and in severe cases, even lead to crop failure. For example, the extremely heavy early winter snowstorm in Hebei in 2009 severely damaged the wheat seedling situation, and the spring freezing injury in 2013 caused large-scale spike deformities. With the global climate change and the frequent occurrence of extreme low-temperature events, the demand for improving the antifreeze traits of wheat has become increasingly urgent.

[0003] Traditional wheat antifreeze breeding mainly relies on field phenotype screening, but this method has significant limitations: on the one hand, the occurrence of freezing injury is accidental, and regional adaptability evaluation requires multi-year and multi-point experiments, resulting in a long breeding cycle and high costs; on the other hand, wheat antifreeze is a complex quantitative trait, and its genetic mechanism involves the coordinated action of multiple genes, making it difficult to accurately aggregate excellent alleles through conventional cross-breeding. In recent years, molecular marker-assisted selection (MAS) technology has provided a new approach for improving antifreeze traits, but the existing marker system still has deficiencies. For example, InDel markers rely on electrophoresis detection, with low throughput and being easily affected by experimental conditions, making it difficult to meet the needs of large-scale breeding.

[0004] KASP (Kompetitive Allele-Specific PCR) marker technology, as a high-throughput and low-cost SNP genotyping tool, has shown significant advantages in crop genetic research. This technology is based on the design of dual-color fluorescent probes and universal primers, and can achieve automated detection on a common quantitative PCR platform, and the genotyping of thousands of samples can be completed in a single reaction. Compared with traditional markers, KASP has three core advantages: firstly, the detection throughput is increased by a hundred times, supporting genome-wide association analysis (GWAS) and high-throughput screening of breeding populations; secondly, closed-tube detection is used to avoid contamination, and the genotyping accuracy exceeds 99%; thirdly, the reagent cost is reduced by more than 60%, significantly reducing the breeding cost.

[0005] Currently, KASP technology has made breakthroughs in the research of wheat disease resistance, quality and other traits. For example, the Saintenac team (2013) used KASP markers to finely map the QTLs for wheat stem rust resistance and successfully cloned three key resistance genes. In the field of cold tolerance traits, although QTLs such as Fr-1 and Fr-2 on chromosome 5A have been preliminarily mapped, there is a lack of KASP markers that can be directly applied to breeding. By integrating genomic sequencing data and wheat phenotype data under low temperature stress, developing closely linked KASP markers will enable the efficient polymerization of cold tolerance genes and break through the genetic bottleneck of traditional breeding.

[0006] In summary, the development of KASP molecular markers related to wheat cold tolerance can not only fill the gaps in existing technologies, but also promote the transformation of wheat cold tolerance breeding from empirical selection to precision design, providing key technical support for coping with climate change challenges and ensuring food security. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide molecular markers for assisting in screening cold-tolerant wheat germplasm and their applications.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0009] A method for screening or assisting in screening wheat with different cold tolerances, comprising the following steps: detecting whether the wheat to be tested is of genotype qFDG-7A.3a or genotype qFDG-7A.3b, and the cold tolerance of genotype qFDG-7A.3a wheat > the cold tolerance of genotype qFDG-7A.3b wheat;

[0010] The wheat of genotype qFDG-7A.3a is wheat with a homozygous AA genotype at the A129C SNP locus;

[0011] The wheat of genotype qFDG-7A.3b is wheat with a homozygous CC genotype at the A129C SNP locus;

[0012] The A129C SNP locus is the 129th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0013] Further preferably, the step of detecting whether the wheat to be tested is of genotype qFDG-7A.3b or genotype qFDG-7A.3a is as follows:

[0014] (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;

[0015] The primer combination consists of the upstream primer F1 shown in SEQ ID NO: 2, the upstream primer F2 shown in SEQ ID NO: 3, and the downstream primer R shown in SEQ ID NO: 4;

[0016] (a2) After completing step (a1), use an instrument to detect the fluorescence signal of the PCR amplification product, and obtain the genotype of the wheat to be tested according to the color of the fluorescence signal.

[0017] Further preferably, the steps for detecting whether the wheat to be tested is genotype qFDG-7A.3b or genotype qFDG-7A.3a are as follows:

[0018] (b1) Using the genomic DNA of the wheat to be tested as a template, perform PCR amplification with the primer combination to obtain a PCR amplification product;

[0019] The primer combination consists of the upstream primer F1 shown in SEQ ID NO: 2, the upstream primer F2 shown in SEQ ID NO: 3, and the downstream primer R shown in SEQ ID NO: 4;

[0020] (b2) Take the PCR amplification product obtained in step (b1) and sequence it;

[0021] (b3) According to the sequencing result obtained in step (b2), obtain the genotype of the wheat to be tested.

[0022] A kit for identifying or assisting in the identification of wheat frost resistance, comprising substances for detecting whether the wheat to be tested is genotype qFDG-7A.3a or genotype qFDG-7A.3b;

[0023] The genotype qFDG-7A.3a is the homozygous AA genotype at the A129C SNP locus;

[0024] The genotype qFDG-7A.3b is the homozygous CC genotype at the A129C SNP locus;

[0025] The A129C SNP locus is the 129th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0026] Further preferably, the substance for detecting whether the genotype of the wheat to be tested is genotype qFDG-7A.3a or genotype qFDG-7A.3b is a primer combination;

[0027] The primer combination consists of the upstream primer F1 shown in SEQ ID NO: 2, the upstream primer F2 shown in SEQ ID NO: 3, and the downstream primer R shown in SEQ ID NO: 4.

[0028] The molecular marker shown in SEQ ID NO: 1.

[0029] Use of the above-mentioned kit or the above-mentioned molecular marker in identifying or assisting in the identification of wheat frost resistance.

[0030] Use of the above-mentioned kit or the above-mentioned molecular marker in screening or assisting in the screening of wheat with different frost resistances.

[0031] Use of the above-mentioned kit or the above-mentioned molecular marker in wheat breeding.

[0032] Use of the primer combination in the directional cultivation or assisted directional cultivation of wheat lines with high frost resistance, wherein the primer combination consists of the upstream primer F1 shown in SEQ ID NO: 2, the upstream primer F2 shown in SEQ ID NO: 3, and the downstream primer R shown in SEQ ID NO: 4.

[0033] The beneficial effects produced by adopting the above technical solution are as follows: The present invention provides the KASP marker Kasp_qFDG-7A.3 for identifying the allelic variations of qFDG-7A.3a and qFDG-7A.3b and its correlation with wheat frost resistance. Applying the KASP marker in the present invention to molecular marker-assisted selection of wheat frost resistance can quickly and efficiently screen out wheat varieties (germplasms) with high frost resistance, thereby accelerating the breeding process of new high-quality wheat varieties. The present invention has important theoretical significance and economic value for using molecular marker-assisted selection to select wheat germplasms or breeding offspring materials with high frost resistance. Brief Description of the Drawings

[0034] Figure 1 It is the KASP primer position of the 129th base from the 5'-end of the sequence shown in SEQ ID NO: 1 for two allelic types of the frost resistance-related QTL qFDG-7A.3 in common wheat of the present invention; the square box represents the 129th base from the 5'-end of the sequence shown in SEQ ID NO: 1, and the positions of the upstream and downstream primers of the KASP marker are identified by double underlines. The sequence in the figure is the sequence of SEQ ID NO: 1;

[0035] Figure 2 It is a schematic diagram of the detection results of the genotyping of 351 wheat germplasms in the natural population. In the figure, AA is the AA homozygous type, that is, the genotype qFDG-7A.3a; CC is the CC homozygous type, that is, the genotype qFDG-7A.3b; CK is the negative control;

[0036] Figure 3Schematic diagram of the correlation analysis results between wheat germplasms with genotype qFDG-7A.3a and genotype qFDG-7A.3b and the mean value of freezing tolerance in natural populations under different environments (“*” indicates P<0.05, that is, the difference reaches a significant level; “**” and “***” indicate P<0.01 and P<0.001 respectively, that is, the difference reaches an extremely significant level; DX_2022, DX_2023, DX_2024 respectively represent sown in Dingxi City, Gansu Province in 2021 - 2022, 2022 - 2023, 2023 - 2024, SJZ_2023, SJZ_2024 respectively represent sown in Shijiazhuang City, Hebei Province in 2022 - 2023, 2023 - 2024, TS_2024 represents sown in Tangshan City, Hebei Province in 2023 - 2024);

[0037] Figure 4 Photos of the process of extracting genomic DNA from wheat leaves;

[0038] Figure 5 Phenotype photos of freezing tolerance of different wheat varieties in Shijiazhuang in 2022 - 2023. Detailed implementation manners

[0039] The following examples illustrate the present invention in detail. All kinds of raw materials and various equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0040] It should be understood that when used in the specification of the present application and the appended claims, the term “comprising” indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their combinations.

[0041] It should also be understood that the term “and / or” used in the specification of the present application and the appended claims refers to any combination and all possible combinations of one or more of the related listed items, and includes these combinations.

[0042] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in one or more embodiments of this application. Thus, statements such as "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments", etc. that appear in different places in this specification do not necessarily all refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized. The terms "comprising", "including", "having" and their variants all mean "including but not limited to", unless otherwise specifically emphasized.

[0043] In addition, in the description of the specification of this application and the appended claims, the terms "first", "second", "third", etc. are only used for distinguishing descriptions and cannot be understood as indicating or implying relative importance.

[0044] Next, the technical solution of the present invention will be clearly and completely described in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0045] Example 1: Detection of different allelic types of QTL qFDG-7A.3 related to regulating wheat freezing tolerance during the overwintering period using the KASP marker Kasp_FDG-7A.3

[0046] Using the KASP marker Kasp_FDG-7A.3 to detect the QTL qFDG-7A.3 related to wheat freezing tolerance during the overwintering period, the different allelic types are divided into the following two steps: PCR amplification and genotyping. The methods used in the examples are all conventional methods unless otherwise specified. The wheat varieties used are well-known varieties, and most of the varieties are recorded in the following reference: "Zhao Jie, Sun Lijing, Gao Huimin, Hu Mengyun, Mu Liming, Cheng Xiaohu, Wang Jianbing, Zhao Yun, Li Qianying, Wang Peinan, Li Hui, Zhang Yingjun. Genome-wide association study of yield-related traits in common wheat (Triticum aestivum L.) under normal and drought treatment conditions, Frontiers in Plant Science, 2023, 13: 1098560". All common wheat varieties are preserved in the Wheat Research Center of the Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences.

[0047] (1) PCR amplification system and procedure. As Figure 4 shown, use TPS solution to extract genomic DNA from wheat leaves, and add 100 ul of ddH2O to dissolve it. Use 1% agarose gel electrophoresis to detect the quality of DNA. The extracted DNA is required to have no obvious impurities, clear bands, and no degradation. After measuring the concentration of DNA, it is uniformly diluted to 28.3 ng / μl, and PCR amplification is carried out using the diluted wheat genomic DNA as a template.

[0048] Preparation of KASP marker primer working solution: Design KASP primers according to the base SNP sequence of the wheat freezing tolerance-related QTL qFDG-7A.3. The polymorphism of this SNP locus is the base difference between C / A. The primer sequences are shown in Table 1. Respectively pipette 12 μl of each of the two upstream primers (100 μM) and 30 μl of the downstream primer (100 μM), and supplement to 100 μl with sterile ultrapure water to serve as the primer working solution for KASP markers, and store at 4°C for later use.

[0049] Table 1 KASP marker primer sequence table for identifying allelic variations of common wheat QTL FDG-7A.3

[0050]

[0051] The PCR amplification system is as follows: 1.5 μl of template DNA, 0.0417 μl of primer working solution, 0.75 μl of 2×KASP Master Mix (LGC, product number: KBS-1050-112), and the reaction system is supplemented to 3 μl with sterile ultrapure water.

[0052] The PCR reaction program is as follows: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s and annealing for 20 s (the first annealing temperature is 61 °C, and the temperature decreases by 0.6 °C for each cycle) for a total of 10 cycles; denaturation at 94 °C for 20 s and annealing at 55 °C for 1 min for a total of 26 cycles; extension at 72 °C for 3 min and preservation at 4 °C.

[0053] (2) Genotyping. After the PCR reaction is completed, a fluorescence signal reader (Omega, BMG LABTECH, Germany) and a fluorescence detection system (Araya) are used to convert the fluorescence signal into an analyzable value to read the fluorescence data of the reaction product. The fluorescence scanning results are graphically displayed using the "ggplot" package in R language. Samples with the A base type carry FAM fluorescence and are distributed near the x-axis; samples with the C base type carry HEX fluorescence and are distributed near the y-axis; samples with no detected signal are distributed near the origin (attached Figure 2 ).

[0054] Example 2: Detection of the freezing injury grade of common wheat varieties during the overwintering period using the KASP marker Kasp_FDG-7A.3

[0055] Among 351 Chinese wheat varieties, 299 varieties are of the allelic type qFDG-7A.3a, and 52 varieties are of the allelic type qFDG-7A.3b. These 351 varieties were sown in Shijiazhuang City, Hebei Province in 2022 - 2023 (the freezing tolerance phenotypes are as Figure 5 shown), sown in Dingxi City, Gansu Province in 2023 - 2024, sown in Tangshan City, Hebei Province in 2023 - 2024, with a row length of 2 m, randomized block design, and each material was planted with 2 to 4 replicates.

[0056] Evaluate the winter freezing injury grade of wheat 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 the freezing injury grade, levels 1, 1+, 2-, 2, 2+, 3-, 3, 3+, 4-, 4, and 4+ are replaced with integer levels 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively. The larger the value, the more severe the freezing injury of the wheat, the more sensitive it is to low temperature, and the weaker its frost resistance. The allele types of wheat germplasm materials and the freezing injury grades of wheat in different years and different locations are shown in Table 2.

[0057] Table 2 Detection results of Kasp qFDG-7A.3 marker in wheat germplasm and data of wheat freezing injury grades in different years and different locations

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Note: NA represents missing data, and wheat varieties with missing data are not included in the statistical analysis of freezing injury grades.

[0071] The results showed that the mean freezing injury grades of wheat varieties carrying the allelic type qFDG-7A.3a were lower than those of wheat varieties carrying the allelic type qFDG-7A.3b in different years and locations, and the difference between the two reached a significant or highly significant level (P<0.01 or P<0.001) (as shown in Table 3, Figure 3 ).

[0072] Table 3 Statistical analysis of the relationship between allelic variation types of QTL qFDG-7A.3 in common wheat and freezing tolerance

[0073]

[0074] Note: The statistical analysis used a two-tailed t-test; * represents a significant difference level, and ** represents a highly significant difference level.

[0075] Through a large number of experiments, the inventors of the present invention found a SNP locus in the wheat genome (Chinese Spring wheat genome IWGSC RefSeqv1.0), named the A129C SNP locus. The A129C SNP locus is located at the 129th position from the 5' end of SEQ ID NO: 1, and the genotypes are AA homozygous and CC homozygous. According to the SNP (A / C) difference locus, a KASP marker Kasp_qFDG-7A.3 for identifying different freezing tolerances was developed. The allele type qFDG-7A.3a (AA homozygous) with FAM fluorescence and distributed near the x-axis is the freezing-tolerant allele of wheat, and the allele type qFDG-7A.3b (CC homozygous) with HEX fluorescence and distributed near the y-axis is the low-temperature sensitive allele. By identifying the phenotypes of 351 Chinese wheat variety resources over many years and at multiple locations during the overwintering period, it was shown that this marker can accurately genotype the two allelic types of qFDG-7A.3a and qFDG-7A.3b. The molecular marker related to wheat freezing tolerance provided by the present invention can be used to detect whether a wheat variety or line contains freezing-tolerant loci, which has high value for wheat freezing tolerance breeding. At the same time, the developed KASP molecular marker can greatly accelerate the breeding process of wheat freezing-tolerant varieties.

[0076] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these examples without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0077] In the above embodiments, the descriptions of each embodiment have their own focuses. For parts not detailed or recorded in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0078] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; 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 invention, and should all be included in the protection scope of the present invention.

Claims

1. A method for screening or assisting in screening wheat with different frost resistance, characterized in that: The method comprises the following steps: detecting whether the wheat to be tested is a genotype qFDG-7A.3a or a genotype qFDG-7A.3b, wherein the freezing resistance of the genotype qFDG-7A.3a wheat is greater than the freezing resistance of the genotype qFDG-7A.3b wheat; The wheat of the genotype qFDG-7A.3a is wheat of which the genotype of the A129C SNP site is AA homozygous; The wheat of the genotype qFDG-7A.3b is wheat whose genotype at the A129C SNP site is CC homozygous; The A129C SNP site is the 129th 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 of detecting whether the wheat to be tested is genotype qFDG-7A.3b or genotype qFDG-7A.3a are as follows: (a1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification 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 completing step (a1), an instrument is used to detect the fluorescent signal of the PCR amplification product, and the genotype of the wheat to be tested is obtained according to the color of the fluorescent signal.

3. The method according to claim 1, characterized in that The steps of detecting whether the wheat to be tested is genotype qFDG-7A.3b or genotype qFDG-7A.3a are as follows: (b1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification 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) taking the PCR amplification product obtained in step (b1) and sequencing it; (b3) Obtaining the genotype of the wheat to be tested according to the sequencing results obtained in step (b2).

4. A kit for identifying or assisting in identifying the frost resistance of wheat, characterized in that: It includes a material for detecting whether the wheat to be tested is of genotype qFDG-7A.3a or genotype qFDG-7A.3b; The genotype qFDG-7A.3a is that the genotype of the A129C SNP site is AA homozygous; The genotype qFDG-7A.3b is that the genotype of the A129C SNP site is CC homozygous; The A129C SNP site is the 129th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

5. The kit according to claim 4, characterized in that The material for detecting whether the genotype of the wheat to be tested is the genotype qFDG-7A.3a or the genotype qFDG-7A.3b 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 according to any one of claims 4 to 5 or the molecular marker according to claim 6 in identifying or assisting in identifying the frost resistance of wheat.

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

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

10. Use of a primer combination in directional breeding or auxiliary directional breeding of wheat lines with high frost resistance, the 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.

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