SNP (Single Nucleotide Polymorphism) molecular marker site related to cold resistance of wheat and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker site related to wheat cold resistance and application of the SNP molecular marker site. The molecular site is located at the 101st basic group from the 5'tail end of a sequence as shown in SEQ ID NO.1, and the polymorphism is C / T. The molecular marker related to wheat cold resistance in the wintering period provided by the invention can be used for detecting whether a wheat variety or strain contains a cold-resistant site or not, and has very high value for cold-resistant breeding of wheat, and meanwhile, the developed KASP molecular marker can be used for greatly accelerating the breeding process of the wheat cold-resistant variety.

Description

Technical Field

[0001] The invention relates to the field of crop genetic breeding, in particular to SNP molecular marker sites related to wheat cold resistance and applications thereof. Background Art

[0002] In recent years, with the intensification of global climate change, the frequency and intensity of extreme low temperatures have also continued to increase, and wheat production has continued to be increasingly affected by low temperature stress. According to statistics, the global wheat production is reduced by millions of tons every year due to extreme low temperatures in winter, and low temperature stress has become one of the important factors affecting wheat production. Therefore, the development of polymorphic molecular markers related to wheat cold resistance will not only provide an effective means for the identification and screening of wheat cold-resistant germplasm resources, but also provide important genetic information for molecular design breeding of wheat cold resistance.

[0003] In recent years, with the development of wheat genome sequencing and assembly strategies, wheat genome sequencing has made a series of major progress, laying a good foundation for the development of wheat molecular markers. KASP (Kompetitive Allele-Specific PCR) markers, namely competitive allele-specific PCR, use FAM or HEX fluorescent groups added to the end of the primers to type the target sequence based on the reading judgment of the PCR terminal fluorescent signal, so as to achieve the purpose of identifying the specific SNP (single base nucleotide polymorphism) or InDel (insertion / deletion) contained in the target allele. Compared with traditional detection technology, its operation process has the advantages of high flexibility, low cost, precision and high throughput, which can accelerate the process of molecular marker-assisted selection and realize efficient and accurate breeding of new crop varieties. Therefore, the development of KASP markers for identifying wheat cold resistance will provide an effective detection method for breeding cold-resistant wheat varieties and ensure my country's food security and sustainable agricultural development. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide SNP molecular marker sites related to wheat cold resistance and applications thereof.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0006] A SNP molecular site related to wheat cold resistance, the molecular site is located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism is C / T.

[0007] A primer combination includes two specific forward primers and a specific reverse primer for amplifying a SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, the nucleotide sequences of the two specific forward primers are shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequence of the specific reverse primer is shown in SEQ ID NO.4.

[0008] A kit for detecting the SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1 comprises the primer combination.

[0009] A SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, a primer combination consisting of two specific forward primers and one specific reverse primer for amplifying the SNP molecular site, and a kit containing the primer combination are used in identifying and screening cold-resistant wheat varieties or lines.

[0010] A SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, a primer combination consisting of two specific forward primers and one specific reverse primer for amplifying the SNP molecular site, and a kit containing the primer combination are used in wheat cold resistance molecular marker assisted breeding.

[0011] A SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, a primer combination consisting of two specific forward primers and one specific reverse primer for amplifying the SNP molecular site, and a kit containing the primer combination are used in improving wheat cold-resistant germplasm resources.

[0012] A SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, a primer combination consisting of two specific forward primers and one specific reverse primer for amplifying the SNP molecular site, and a kit containing the primer combination are used in detecting the cold resistance of wheat during wintering.

[0013] A method for detecting cold resistance of wheat using a SNP molecular site located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, characterized in that the method comprises the following steps:

[0014] a. Extracting genomic DNA of the sample to be tested from wheat leaves;

[0015] b. Using genomic DNA as a template, performing PCR amplification on the target sequence in the genomic DNA of the sample to be tested using the primer combination as described in claim 2 or the kit as described in claim 3;

[0016] c. The amplified samples are tested using a fluorescence detection platform, and the cold resistance of wheat is determined based on the polymorphism of the SNP molecular markers reflected by the obtained fluorescence signals;

[0017] d. If the amplification result shows that the base mutation of the SNP molecular marker in claim 1 is T, the tested wheat sample has weak cold resistance; if the polymorphism of the SNP molecular marker is C, the tested wheat sample has strong cold resistance.

[0018] Further preferably, the step a specifically comprises using TPS solution to extract wheat leaf genomic DNA, adding 100ul ddH2O to dissolve; using 1% agarose gel electrophoresis to detect DNA quality, and the extracted DNA is required to have no obvious impurities, clear bands, and no degradation; after the DNA concentration is measured, it is uniformly diluted to 28.3ng / μl, and the diluted wheat genomic DNA is used as a template.

[0019] Further preferably, the step c is specifically to use a fluorescent signal reader (Omega, BMGLABTECH, Germany) and a fluorescent detection system (Araya) to convert the fluorescent signal into an analyzable numerical value after the PCR reaction is completed to read the fluorescent data of the reaction product, and the fluorescence scanning result is graphically displayed using the R language "ggplot" package, the C base type has FAM fluorescence, and the T base type has HEX fluorescence.

[0020] The beneficial effect of adopting the above technical scheme is that the molecular markers related to the cold resistance of wheat during the wintering period provided by the research group of the present invention can be used to detect whether wheat varieties or strains contain cold-resistant sites, which is of great value for the cold-resistant breeding of wheat. At the same time, the use of the developed KASP molecular markers can greatly accelerate the breeding process of cold-resistant wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the KASP primer position of the two allele types of the common wheat cold resistance related QTL qFDG-5A.2 in the present invention at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1; the box represents 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 marked with double underlines. The sequence in the figure is the sequence of SEQ ID NO.1.

[0022] Figure 2 This is a diagram of the marker detection results of the wheat variety Kasp_FDG-5A.2 of the present invention. DETAILED DESCRIPTION

[0023] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained by purchasing from the market. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0024] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of 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 combinations thereof.

[0025] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0026] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0027] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0028] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention develops a KASP marker Kasp_FDG-5A.2 for detecting wintering cold resistance of wheat based on the 101st base from the 5' end of the sequence shown in SEQ ID NO.1 of the common wheat cold resistance related site, and the SNP (C / T) difference site contained in the two allele types qFDG-5A.2a and qFDG-5A.2b of the wheat variety Chinese Spring genome IWGSC_RefSeq_v1.0, http: / / 202.194.139.32 / jbrowse-1.12.3-release / ). The type of wheat cold resistance (qFDG-5A.2a) carrying FAM fluorescence and distributed near the x-axis, and the type of wheat low temperature sensitivity (qFDG-5A.2b) carrying HEX fluorescence and distributed near the y-axis. By identifying the multi-year and multi-point wintering phenotypes of 403 Chinese wheat variety resources (see Table 1), it was shown that the marker can accurately type the two allele types, qFDG-5A.2a and qFDG-5A.2b.

[0030] Example 1 Using KASP marker Kasp_FDG-5A.2 to detect different allele types of QTL qFDG-5A.2 related to regulating wintering resistance of wheat

[0031] The KASP marker Kasp_FDG-5A.2 was used to detect the different allele types of QTL qFDG-5A.2 at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, which was related to regulating the cold resistance of wheat during the wintering period. The steps were as follows: PCR amplification and genotyping.

[0032] (1) PCR amplification system and procedure. Wheat leaf genomic DNA was extracted using TPS solution and dissolved by adding 100ul ddH2O. DNA quality was tested by 1% agarose gel electrophoresis. The extracted DNA was required to be free of obvious impurities, with clear bands and no degradation. After the DNA concentration was measured, it was uniformly diluted to 28.3ng / μl, and PCR amplification was performed using the diluted wheat genomic DNA as a template.

[0033] Preparation of KASP marker primer working solution: KASP primers were designed based on the SNP of the 101st base from the 5' end of the sequence shown in SEQ ID NO.1 of the wheat cold resistance related QTL qFDG-5A.2. The polymorphism of the SNP site is a C / T base difference. The primer sequences are shown in Table 1 and Figure 1 . Take 12 μl of each of the two upstream primers (primer A, primer B, 100 μM) and 30 μl of the downstream primer (primer C, 100 μM), add sterile ultrapure water to 100 μl as the KASP-labeled primer working solution, and store at 4°C for later use.

[0034] Table 1 Primer sequences of KASP marker Kasp_FDG-5A.2 for detecting cold resistance of wheat

[0035]

[0036] The PCR amplification system was 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 was supplemented to 3 μl with sterile ultrapure water.

[0037] The PCR reaction program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing for 20 s (the first annealing temperature was 61°C, and the temperature was lowered by 0.6°C each cycle) for a total of 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 1 min for a total of 26 cycles; extension at 72°C for 3 min, and storage at 4°C.

[0038] (2) Genotyping. After the PCR reaction was completed, the fluorescence signal was converted into an analyzable value using a fluorescence signal reader (Omega, BMG LABTECH, Germany) and a fluorescence detection system (Araya) to read the fluorescence data of the reaction product. The fluorescence scanning results were graphically displayed using the R language "ggplot" package. The C base type has FAM fluorescence, which is distributed near the x-axis; the T base type has HEX fluorescence, which is distributed near the y-axis; samples with no detected signal are distributed near the origin (see Figure 2 ).

[0039] Example 2: Using KASP marker Kasp_FDG-5A.2 to detect the frost damage level of common wheat varieties during winter

[0040] As shown in Tables 2 and 3, of the 403 Chinese wheat varieties, 307 varieties were of the allele type qFDG-5A.2a and 96 varieties were of the allele type qFDG-5A.2b. These 403 varieties were sown in Shijiazhuang, Hebei Province in 2020-2021, 2022-2023, and 2023-2024, in Dingxi, Gansu Province in 2021-2022, 2022-2023, and 2023-2024, and in Tangshan, Hebei Province in 2023-2024, with a 2-meter row length, a randomized block design, and 2 to 4 replicates per material.

[0041] Table 2 Statistical analysis results of the relationship between allele variation types and freezing damage grades of common wheat QTLqFDG-5A.2

[0042]

[0043] Note: Statistical analysis was performed using a two-tailed t-test; * indicates a significant difference, and ** indicates an extremely significant difference.

[0044] Table 3 Detection results of wheat cold resistance marker Kasp_FDG-5A.2KASP and freezing damage grade data

[0045]

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[0059] Note: NA represents missing data. The frost damage level of wheat varieties with missing data is not counted.

[0060] For the statistical analysis of frost damage levels, levels 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 serious the frost damage to the wheat, the more sensitive it is to low temperatures, and the weaker its cold resistance.

[0061] The results showed that the mean frost damage levels of wheat varieties carrying the allele qFDG-5A.2a in different years and locations were lower than those of wheat varieties carrying the allele qFDG-5A.2b, and the two were significantly or extremely significantly different (P<0.05 or P<0.01).

[0062] The molecular markers related to wintering cold resistance of wheat provided by the present invention can be used to detect whether wheat varieties or strains contain cold resistance sites, which is of great value for cold resistance breeding of wheat. At the same time, the developed KASP molecular markers can greatly accelerate the breeding process of cold-resistant wheat varieties.

[0063] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

[0064] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0065] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions 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. The SNP molecular loci related to wheat cold resistance are characterized by: The molecular site is located at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1, and the polymorphism is C / T.

2. A primer combination, characterized in that: It comprises two specific forward primers and one specific reverse primer for amplifying the SNP molecular site described in claim 1, the nucleotide sequences of the two specific forward primers are shown as SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequence of the specific reverse primer is shown as SEQ ID NO.

4.

3. A kit for detecting the SNP molecular site according to claim 1, characterized in that: Comprising the primer combination of claim 2.

4. Use of the SNP molecular locus according to claim 1, the primer combination according to claim 2, and the kit according to claim 3 in identifying and screening cold-resistant wheat varieties or strains.

5. Use of the SNP molecular locus described in claim 1, the primer combination described in claim 2, and the kit described in claim 3 in molecular marker-assisted breeding for cold resistance of wheat.

6. Use of the SNP molecular locus described in claim 1, the primer combination described in claim 2, and the kit described in claim 3 in improving cold-resistant wheat germplasm resources.

7. Use of the SNP molecular locus described in claim 1, the primer combination described in claim 2, and the kit described in claim 3 in detecting the cold resistance of wheat during the wintering period.

8. The method for detecting cold resistance of wheat using the SNP molecular loci according to claim 1, characterized in that: The method comprises the following steps: Extracting genomic DNA of the sample to be tested from wheat leaves; Using genomic DNA as a template, using the primer combination as described in claim 2 or the kit as described in claim 3 to perform PCR amplification on the target sequence in the genomic DNA of the sample to be tested; The amplified samples were tested using a fluorescence detection platform, and the cold resistance of wheat was determined based on the polymorphism of the SNP molecular markers reflected by the obtained fluorescence signals. If the amplification result shows that the base mutation of the SNP molecular marker in claim 1 is T, the cold resistance of the wheat sample to be tested is weak; if the polymorphism of the SNP molecular marker is C, the cold resistance of the wheat sample to be tested is strong.

9. The method for detecting cold resistance of wheat using SNP molecular loci according to claim 8, characterized in that: The step a specifically comprises extracting wheat leaf genomic DNA using TPS solution, adding 100 ul ddH2O to dissolve it; performing DNA quality detection using 1% agarose gel electrophoresis, wherein the extracted DNA is required to have no obvious impurities, clear bands, and no degradation; after measuring the DNA concentration, the DNA is uniformly diluted to 28.3 ng / μl, and the diluted wheat genomic DNA is used as a template.

10. The method for detecting cold resistance of wheat using SNP molecular loci according to claim 8, characterized in that: The step c specifically includes converting the fluorescence signal into an analyzable numerical value using a fluorescence signal reader (Omega, BMG LABTECH, Germany) and a fluorescence detection system (Araya) after the PCR reaction is completed to read the fluorescence data of the reaction product. The fluorescence scanning results are graphically displayed using the R language "ggplot" package. The C base type has FAM fluorescence, and the T base type has HEX fluorescence.

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