SNP molecular marker loci related to wheat cold resistance and their applications

Abstract

The present invention discloses a SNP molecular marker site associated with wheat cold resistance and its application. The molecular marker site is located at the 101st base from the 5' end of the sequence shown in SEQ ID NO. 1 and has a C / T polymorphism. The molecular marker associated with wheat cold resistance during wintering provided by the present invention can be used to detect whether wheat varieties or strains contain the cold resistance site, which is of great value for wheat cold resistance breeding. Furthermore, the use of the developed KASP molecular marker can greatly accelerate the breeding process of cold-resistant wheat varieties.

Description

Technical Field

[0001] The present 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 cold temperatures have continued to increase, and the impact of cold stress on wheat production has continued to grow. According to statistics, global wheat production is reduced by millions of tons each year due to extreme winter temperatures, making cold stress a major factor affecting wheat production. Therefore, the development of polymorphic molecular markers associated with cold resistance in wheat will not only provide an effective means for the identification and screening of cold-resistant wheat germplasm resources, but also provide important genetic information for molecular design breeding of cold-resistant wheat varieties.

[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 to add to the end of the primers, and 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 technologies, 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 the 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, ensuring 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 comprises two specific forward primers and one 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, wherein 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 the wintering period.

[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, PCR amplification of the target sequence in the genomic DNA of the test sample is performed using the primer combination as described in claim 2 or the kit as described in claim 3;

[0016] c. The amplified samples were tested using a fluorescence detection platform, and the cold resistance of the wheat was 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, step a specifically comprises extracting wheat leaf genomic DNA using TPS solution and adding 100ul ddH2O to dissolve it; performing DNA quality detection using 1% agarose gel electrophoresis, and the extracted DNA is required to be free of obvious impurities, clear bands, and non-degraded; after measuring the DNA concentration, uniformly diluting it to 28.3ng / μl, and using the diluted wheat genomic DNA as a template.

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

[0020] The beneficial effect of adopting the above technical solution is that the molecular markers related to wheat cold resistance during the wintering period provided by the research team of the present invention can be used to detect whether wheat varieties or strains contain cold-resistant sites, which is of great value for wheat cold-resistant breeding. 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 The KASP primer positions for the two allelic forms of the QTL qFDG-5A.2 associated with cold resistance in common wheat (SEQ ID NO. 1) are shown. The box represents the 101st base from the 5' end of the sequence shown in SEQ ID NO. 1. The positions of the primers upstream and downstream of the KASP marker are double underlined. The sequence in the figure is that of SEQ ID NO. 1.

[0022] Figure 2 This is a diagram showing the results of marker detection for 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 through commercial purchase. The experimental methods used in the following examples are all 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, 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 collections thereof.

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

[0026] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in other embodiments" appearing in various places in 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 "including," "comprising," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized.

[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 solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Based on the 101st base from the 5' end of the common wheat cold resistance locus in the sequence shown in SEQ ID NO. 1, and referring to the SNP (C / T) differential sites contained in the two allelic 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 / ), a KASP marker, Kasp_FDG-5A.2, was developed for detecting wintering cold resistance in wheat. The FAM-fluorescent alleles located near the x-axis indicate the cold-resistant allele (qFDG-5A.2a), while the HEX-fluorescent alleles located near the y-axis indicate the cold-sensitive allele (qFDG-5A.2b). 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 allelic types qFDG-5A.2a and qFDG-5A.2b.

[0030] Example 1: Application of KASP marker Kasp_FDG-5A.2 to detect different alleles of QTL qFDG-5A.2 regulating wintering resistance of wheat

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

[0032] (1) PCR amplification system and procedure. Wheat leaf genomic DNA was extracted using TPS solution and dissolved in 100 μl of ddH2O. DNA quality was tested using 1% agarose gel electrophoresis. The extracted DNA was required to be free of obvious impurities, with clear bands and no degradation. After DNA concentration was measured, it was uniformly diluted to 28.3 ng / μl. 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 at the 101st base from the 5' end of the sequence shown in SEQ ID NO.1 of the wheat cold resistance QTL qFDG-5A.2. The polymorphism of this SNP site is a C / T base difference. The primer sequences are shown in Table 1 and Figure 1 12 μl of each of the two upstream primers (primer A and primer B, 100 μM) and 30 μl of the downstream primer (primer C, 100 μM) were taken separately, and the volume was made up to 100 μl with sterile ultrapure water. This was used as the KASP-labeled primer working solution and stored at 4°C for later use.

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

[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 numerical 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 had FAM fluorescence and was distributed near the x-axis; the T base type had HEX fluorescence and was distributed near the y-axis; samples with no detection signal were distributed near the origin (see Figure 2 ).

[0039] Example 2: Application of KASP marker Kasp_FDG-5A.2 to detect the degree of frost damage of common wheat varieties during wintering

[0040] As shown in Tables 2 and 3, of the 403 Chinese wheat varieties, 307 were of the qFDG-5A.2a allelic type and 96 were of the qFDG-5A.2b allelic type. 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. A randomized block design was used with 2-meter row lengths 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 significant differences, and ** indicates extremely significant differences.

[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. Wheat varieties with missing data are not included in the statistics of frost damage grades.

[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 grade of wheat varieties carrying the allele type qFDG-5A.2a in different years and locations was lower than that of wheat varieties carrying the allele type qFDG-5A.2b, and the difference between the two was significant or extremely significant (P<0.05 or P<0.01).

[0062] The molecular markers related to wheat cold resistance during the wintering period 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 wheat cold resistance breeding. At the same time, the use of 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 various changes, modifications, substitutions and variations may 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.

[0064] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description 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 they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. A primer combination, characterized in that: It includes two specific forward primers and one specific reverse primer for amplifying the SNP molecular site. The SNP 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. 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.

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

3. Use of the primer combination according to claim 1 and the kit according to claim 2 in identifying and screening cold-resistant wheat varieties or lines.

4. Use of the primer combination according to claim 1 and the kit according to claim 2 in molecular marker-assisted breeding for cold resistance in wheat.

5. Use of the primer combination according to claim 1 and the kit according to claim 2 in improving cold-resistant wheat germplasm resources.

6. Use of the primer combination according to claim 1 and the kit according to claim 2 in detecting the cold resistance of wheat during the wintering period.

7. A method for detecting cold resistance of wheat using the primer combination according to claim 1 or the kit according to claim 2, characterized in that: The method comprises the following steps: a. Extracting genomic DNA of the sample to be tested from wheat leaves; b. Using genomic DNA as a template, PCR amplification of the target sequence in the genomic DNA of the test sample is performed using the primer combination according to claim 1 or the kit according to claim 2; c. The amplified samples were tested using a fluorescence detection platform, and the cold resistance of the wheat was determined based on the polymorphism of the SNP molecular markers reflected by the obtained fluorescence signals; 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.

8. The method for detecting cold resistance of wheat using the primer combination according to claim 1 or the kit according to claim 2 according to claim 7, characterized in that: Step a specifically comprises extracting wheat leaf genomic DNA using TPS solution and dissolving it by adding 100 μl of ddH2O; performing DNA quality testing using 1% agarose gel electrophoresis, wherein the extracted DNA is required to be free of obvious impurities, have clear bands, and be free of degradation; and after measuring the DNA concentration, uniformly diluting it to 28.3 ng / μl, and using the diluted wheat genomic DNA as a template.

9. The method for detecting cold resistance of wheat using the primer combination according to claim 1 or the kit according to claim 2 according to claim 7, characterized in that: Specifically, step c comprises converting the fluorescence signal into an analyzable numerical value after the PCR reaction is completed using a fluorescence signal reader and a fluorescence detection system to read the fluorescence data of the reaction product. The fluorescence scanning results are graphically displayed using the R language "ggplot" package, where the C base type is represented by FAM fluorescence and the T base type is represented by HEX fluorescence.

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