Molecular marker and specific primer for detecting main-effect genetic locus qgpc.caas-7al of kernel protein content

Abstract

The application discloses a molecular marker and specific primers for detecting a kernel protein content major-effect genetic locus QGPC.caas-7AL. The application belongs to the technical field of biology and particularly relates to a molecular marker and specific primers for detecting a kernel protein content major-effect genetic locus QGPC.caas-7AL. The SNP site in the application is a SNP site on a wheat 7A chromosome, and the nucleotide type is C or T, which is the 36th nucleotide in sequence 1 in the sequence table. The substance for detecting the polymorphism or genotype of the SNP in the wheat genome can be applied to identifying or assisting in identifying the kernel protein content of wheat and wheat breeding. The substance for detecting the polymorphism or genotype of the SNP site can be combined with other substances (such as a substance for detecting the single nucleotide polymorphism or genotype of other molecular markers related to the kernel protein content of wheat) to prepare a product for identifying high kernel protein content of wheat.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to molecular markers and specific primers for detecting the major genetic locus QGPC.caas-7AL of seed protein content. Background Technology

[0002] Wheat is one of the world's three major food crops, and improving its processing quality is an important breeding goal. The main traits affecting processing quality include grain protein content and quality, starch quality, and color. Specific requirements are related to the type of food and processing method, but protein content is a key target for improving processing quality both domestically and internationally.

[0003] Protein content is significantly influenced by variety, environment, and their interactions, with environmental effects being relatively minor, while variety and its interaction with the environment have a greater impact. Improving grain protein content is an important breeding objective for enhancing the nutritional and processing quality of wheat. Therefore, the discovery and utilization of genetic loci related to grain protein content are crucial for breeding high-quality wheat varieties.

[0004] KASP markers have been widely used to detect SNP loci in crops such as wheat, rice, and maize, enabling high-throughput genotyping without the need for electrophoresis. Using wheat SNP microarray genotyping data for QTL mapping and genome-wide association analysis, linked SNPs can be converted into KASP markers, which can then be directly applied to marker-assisted selection breeding.

[0005] Jimai 22 is a high-yielding, multi-resistant, and high-quality medium-gluten wheat variety, approved by Shandong Province and the National Huang-Huai-Bei Region in September 2006 and January 2007, respectively. It is suitable for planting in the Huai-Bei region and the northern part of the Huang-Huai winter wheat region. Zhongmai 578 is a high-yielding, multi-resistant, and high-quality strong-gluten wheat variety, approved by the National Huang-Huai-Bei Region in June 2021. It is suitable for planting in the irrigated areas of the northern part of the Huang-Huai winter wheat region, including all of Shandong Province, the southern part of Baoding and Cangzhou in Hebei Province, and the basin irrigation areas of Yuncheng and Linfen in Shanxi Province. Compared with Jimai 22, Zhongmai 578 has a higher grain protein content. This invention uses Zhongmai 578 and Jimai 22 to construct a recombinant inbred line (RIL) population including 262 families to study molecular markers related to grain protein content. Summary of the Invention

[0006] The problem this invention aims to solve is how to identify or assist in identifying the protein content of wheat grains and to carry out wheat breeding.

[0007] To address the above technical problems, this invention first provides the application of a substance for detecting KASP polymorphisms or genotypes in the wheat genome in any of the following:

[0008] (1) To identify or assist in the identification of wheat grain protein content;

[0009] (2) Wheat breeding;

[0010] (3) Prepare products for identification or auxiliary identification of wheat grain protein content;

[0011] (4) Prepare products for wheat breeding;

[0012] The SNP site is a site on wheat chromosome 7A, and its nucleotide type is C or T, which is the 36th nucleotide of sequence 1 in the sequence listing.

[0013] The genome sequence of the common wheat variety Chinese Spring (IWGSC_RefSeq_v1.0) (https: / / urgi.versailles.inra.fr / jbrowseiwgsc / gmod_jbrowse / ?data=myData / IWGSC_RefSeq_v1.0) was used as the reference genome. The SNP site was located at 675500189 bp on wheat chromosome 7A (specifically, position 36 of sequence 1 in the sequence listing).

[0014] The present invention also provides a method for identifying or assisting in the identification of wheat grain protein content, comprising detecting the genotype of the SNP locus in the genome of the wheat to be tested, and identifying or assisting in the identification of wheat grain protein content based on the genotype, wherein the genotype is CC or TT, wherein CC is a homozygous type of the SNP being C, and TT is a homozygous type of the SNP being T.

[0015] As one implementation scheme, the method for identifying or assisting in the identification of wheat grain protein content may include the following steps:

[0016] (1) Using the genomic DNA of the wheat to be tested as a template, KASP molecular marker detection was performed using a primer composition; the primer composition consisted of primer A, primer B and primer C;

[0017] Primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-41 of sequence 2 in the sequence listing;

[0018] Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-41 of sequence 3 in the sequence listing.

[0019] Primer C is a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence listing;

[0020] (2) After completing step (1), perform fluorescence detection to determine the genotype of the SNP in the wheat to be tested;

[0021] (3) Identify the grain protein content of the wheat to be tested based on the genotype results: The grain protein content of the wheat to be tested with the SNP genotype TT is better than that of the wheat to be tested with the SNP genotype CC.

[0022] This invention also provides a method for wheat breeding.

[0023] The wheat breeding method provided by the present invention includes detecting the genotype of the SNP locus in the wheat genome, selecting wheat with the genotype TT of the SNP as the parent for breeding, wherein TT is the homozygous type of the SNP being T.

[0024] As an implementation method, wheat breeding methods may include the following steps:

[0025] (1) Using the genomic DNA of the wheat to be tested as a template, the above primer set was used to detect KASP molecular markers;

[0026] (2) After completing step (1), perform fluorescence detection to determine the genotype of the SNP site in the wheat to be tested;

[0027] (3) Select TT genotype wheat as a wheat breed with high grain protein content.

[0028] In the above method, the primer dissolution and preparation method can be as follows: First, dilute the three primers to 100 μM with ddH2O, and then prepare the primer working solution as follows: 12 μL of primer A, 12 μL of primer B, 30 μL of primer C, and 46 μL of ddH2O. This solution is used as the KASP-labeled primer working solution and stored at -20℃ for later use.

[0029] In the above method, the KASP reaction system can be as follows: 1.0 μL template DNA, 0.0336 μL primer working solution, 1.5 μL 2×KASP Master Mix (LGC, Lot No. 13426773), and the reaction system can be made up to 3 μL with sterile ultrapure water.

[0030] In the above method, KASP labeling can be performed on a 384-well PCR instrument (BIO-RAD, S1000TM Thermal Cycler).

[0031] In the above method, the reaction procedure for KASP tags can be:

[0032] Step 1: Pre-denaturation at 94℃ for 15 min;

[0033] Step 2: Denaturation at 94℃ for 20 seconds, annealing for 20 seconds (the first annealing temperature is 65℃, and the temperature is reduced by 1℃ for each cycle) for a total of 10 cycles; denaturation at 94℃ for 20 seconds, annealing at 55℃ for 1 minute for a total of 32 cycles;

[0034] Step 3: Extend at 72℃ for 10 minutes, then store at 4℃.

[0035] The method described above for determining the genotype of the SNP in the wheat sample is as follows: After the PCR reaction, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) are used to convert the fluorescence signal into analyzable values ​​to read the fluorescence data of the reaction products. Genotyping is performed by reading the fluorescence values ​​at the terminal ends. The fluorescence scanning results are graphically displayed using the R software package. C-base types exhibit FAM fluorescence and are distributed near the x-axis; T-base types exhibit HEX fluorescence and are distributed near the y-axis; samples with no detected signal are distributed near the origin.

[0036] The application of the above methods in wheat breeding also falls within the scope of protection of this invention.

[0037] This invention also provides products for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

[0038] The product provided by this invention for detecting polymorphisms or genotypes of SNP sites in the wheat genome contains any of the aforementioned substances for detecting polymorphisms or genotypes of SNP sites in the wheat genome.

[0039] C1) Products that detect single nucleotide polymorphisms or genotypes related to wheat grain protein content;

[0040] C2) Products used for identifying or assisting in the identification of wheat grain protein content;

[0041] C3) Products used in wheat breeding.

[0042] In the above applications, methods, and products, the substance may be a reagent and / or instrument required to determine the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chips. The SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on one-step reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

[0043] Optionally, the substance may be D1), D2), or D3):

[0044] D1) The substance described is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites;

[0045] D2) The substance described is a PCR reagent containing the primer composition described in D1);

[0046] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0047] Optionally, the amplification may be PCR amplification. The primer composition consists of primer A, primer B, and primer C.

[0048] The kit described in D3 may also include KASP Master Mix.

[0049] In the above applications, methods, and products, the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading). The primer composition described herein may be a primer composition consisting of single-stranded DNA with nucleotide sequences of positions 22-41 of sequence 2 in the sequence listing, single-stranded DNA with nucleotide sequences of positions 22-41 of sequence 3 in the sequence listing, and single-stranded DNA with nucleotide sequences of sequence 4 in the sequence listing. Alternatively, the primer composition may be a primer set consisting of single-stranded DNA shown in sequence 2, sequence 3, and sequence 4 in the sequence listing. Sequence 2 in the sequence listing consists of 41 nucleotides, with nucleotides 1-21 being the FAM adapter sequence (as a marker) and nucleotides 22-41 being the specific sequence; Sequence 3 in the sequence listing consists of 41 nucleotides, with nucleotides 1-21 being the HEX adapter sequence (as a marker) and nucleotides 22-41 being the specific sequence.

[0050] The present invention also provides a DNA molecule, the nucleotide sequence of which is shown in Sequence 1 of the sequence listing.

[0051] The applications of the aforementioned DNA molecules also fall within the scope of protection of this invention. Specifically, the applications are those found in any of the following:

[0052] (1) To identify or assist in the identification of wheat grain protein content;

[0053] (2) Wheat breeding;

[0054] (3) Prepare products for identification or auxiliary identification of wheat grain protein content;

[0055] (4) Prepare products for wheat breeding.

[0056] Optionally, in the above applications, the DNA molecule serves as a detection target.

[0057] The substance that detects the SNP site polymorphism and genotype can be combined with other substances (such as substances that detect single nucleotide polymorphisms or genotypes of other molecular markers related to wheat grain protein content) to prepare a product for identifying wheat varieties with high wheat grain protein content.

[0058] In this article, the breeding objectives may include breeding wheat with high grain protein content.

[0059] In this article, the wheat referred to can be a wheat inbred line or a hybrid offspring of two wheat inbred lines, such as a hybrid offspring of wheat Zhongmai 578 and Jimai 22. The wheat can also be a pure line.

[0060] This invention utilizes QTL analysis to locate a QTL locus, QTLQGPC.caas-7AL, significantly associated with wheat grain protein content. This locus is located on wheat chromosome 7A at a physical location of 670.33–675.50 Mb (referencing the wheat variety *Chinese Spring* genome IWGSC_RefSeq_v1.0), with the right-hand linkage microarray marker AX-109534708. This SNP locus exhibits C / T base differences, and the two allele types are named QTLQGPC.caas-7ALa and QTLQGPC.caas-7ALb, respectively. The type carrying FAM fluorescence and distributed near the x-axis indicates low grain protein content (QTLQGPC.caas-7ALa); the type carrying HEX fluorescence and distributed near the y-axis indicates high wheat grain protein content (QTLQGPC.caas-7ALb). The marker was validated using two groups of 163 Chinese wheat germplasm resources. The results showed that the marker can accurately genotype the two allelic types, QTLQGPC.caas-7ALa and QTLQGPC.caas-7ALb, and can be used for marker-assisted breeding. Attached Figure Description

[0061] Figure 1 The QGPC.caas-7AL curve is located in the Zhongmai 578 × Jimai 22RIL population.

[0062] Figure 2 Genotyping results of 163 wheat varieties using the KASP marker KaspAX-109534708. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0065] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0066] The Zhongmai 578 and Jimai 22 mentioned in the following examples have been described in: Liu D, Zhao D, Zeng J, Sani SR, Tong J, Li M, Li F, Zhou S, Hu W, Xia X, Tian Y, Zhu Q, Wang C, Wang D, He Z, Liu J, Zhang Y. Identification of genetic loci for grain yield-related traits in the wheat population Zhongmai 578 / Jimai 22. Journal of Integrative Agriculture. 2023, 22: 1985-1999. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention; it may not be used for any other purpose.

[0067] The 163 wheat varieties in the following examples have been described in: Li F, Wen W, Liu J, Zhang Y, Cao S, He Z, Rasheed A, Jin H, Zhang C, Yan J, Zhang P, Wan Y, Xia X. Genetic architecture of grain yield in bread wheat based on genome-wide association studies. BMC Plant Biology 2019, 19:168. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention; it may not be used for any other purpose.

[0068] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean values. One-way ANOVA was used, and P < 0.05 (*) indicates a significant difference.

[0069] Example 1: Discovery of QTLs related to grain protein content and acquisition of their KASP markers

[0070] I. Phenotypic Study of Grain Protein Content

[0071] A recombinant inbred line (RIL) population comprising 262 families was constructed using Zhongmai 578 and Jimai 22. The Zhongmai 578 / Jimai 22 RIL population was planted in Xinxiang, Shangqiu, and Luoyang in Henan Province and Gaoyi in Hebei Province during the 2020-2021 season. A completely randomized block design with three replicates was used, with single-row plots, row lengths of 1 m, row widths of 0.25 m, and 30 seeds evenly sown per row. Conventional management was implemented. After maturity, the grains were harvested, and the grain protein content was determined using a near-infrared spectroscopy analyzer (DA 7250, Perten). The specific detection method followed the national standard GB / T 5506.4-2008: protein content (14% wet basis) = protein (dry basis) content × (1-14%).

[0072] Genomic DNA was extracted from the young leaves of the above 262 families using a modified CTAB method, and then analyzed using NanoDrop. TM DNA concentration was determined using a 2000c spectrophotometer, and the DNA sample was adjusted to a standard concentration of 50 ng / μL. DNA quality was then assessed using a 0.8% agarose gel electrophoresis. DNA samples meeting quality standards were subjected to SNP genotyping. SNP analysis was performed using a 50K SNP chip developed in collaboration between the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and Affymetrix Axiom.

[0073] II. Linkage Graph Construction

[0074] The 50K SNP chip contained 54,680 markers, of which 11,526 differed between parents. After removing markers that were heterozygous or had a deletion rate greater than 10%, 10,631 markers remained. Redundant markers were removed using IciMapping 4.1bin, leaving 9,354 markers. These 9,354 markers were imported into the online tool MSTMap, and the SingleGL parameter was selected to construct a large linkage group. Then, based on the genetic distance and chromosomal location information between markers, 34 linkage groups were constructed, containing a total of 1,507 markers.

[0075] III. QTL Analysis

[0076] The IciMapping 4.1 ICIM-ADD method was used to perform QTL analysis on the grain protein content at four environmental conditions and the best linear unbiased estimate (Blue) value. A LOD value of 2.5 was selected. A stable QTL was located on chromosome 7AL and named QGPC.caas-7AL. Figure 1 The most closely linked lateral wing markers were AX-110942203 and AX-109534708, with a physical interval of 670.33 Mb–675.50 Mb. Under different environmental conditions, they explained 4.7–10.2% of the phenotypic variation (Table 1). Figure 1 The SNP site AX-109534708 is position 36 of sequence 1, and its nucleotide type is C or T. The nucleotide sequence of sequence 1 is the sequence at physical location 675500154bp-675500224bp on chromosome 7A of wheat IWGSC Refseq v1.0. In the sequence listing, y in sequence 1 represents c or t. The flanking marker AX-109534708 was converted to KaspAX-109534708 for marker-assisted selection breeding.

[0077] Table 1. Genetic loci for grain protein content in the Zhongmai 578 × Jimai 22RIL population detected by composite interval mapping method. (QGPC.caas-7AL)

[0078]

[0079]

[0080] IV. Obtaining the identification primer set for the KASP marker KaspAX-109534708

[0081] A primer set for detecting SNP polymorphic sites (i.e., the KASP marker KapmaxAX-109534708) based on KASP technology was designed, referred to as the KASP primer set. The KASP primer set consists of two upstream primers (primer A and primer B) and one downstream primer (primer C), and the specific sequences are shown in Table 3.

[0082] Table 2. Sequence information of molecular marker AX-109534708

[0083]

[0084] Table 3. KASP primer information for detecting grain protein content QTLQGPC.caas-7AL

[0085]

[0086] Primer A is a primer with a FAM fluorescent tag sequence (underlined bases) at the 5' end, and primer C amplifies the C fragment of SNP site AX-109534708. The fluorescent signal of the FAM group can be read using an ELISA reader or a real-time PCR instrument.

[0087] Primer B is a primer with a HEX fluorescent tag sequence (underlined bases) at the 5' end, and primer C amplifies the T fragment at the SNP site AX-109534708. The fluorescent signal of the HEX group can be read using an ELISA reader or a real-time PCR instrument.

[0088] The KASP marker KaspAX-109534708 was used to detect different allelic types of wheat grain protein-related QGPC.caas-7AL at physical location 675508361 bp on chromosome 7A (SNP site AX-109534708). This SNP site AX-109534708 represents a C / T base difference, and the two allelic types were named QTLQGPC.caas-7ALa and QTLQGPC.caas-7ALb, respectively. The allelic type carrying FAM fluorescence and located near the x-axis is QTLQGPC.caas-7Ala, with the SNP site containing C nucleotides; the allelic type carrying HEX fluorescence and located near the y-axis is QTLQGPC.caas-7Alb, with the SNP site containing T nucleotides.

[0089] 1. KASP amplification system and procedure

[0090] Preparation of KASP-labeled primer working solution: 12 μL of primer A (100 μmol / L), 12 μL of primer B (100 μmol / L), 30 μL of primer C (100 μmol / L), and 46 μL of ddH2O were used as the KASP-labeled primer working solution and stored at -20℃ for later use.

[0091] The PCR amplification system consisted of: 1.0 μL template DNA, 0.0336 μL primer working solution, 1.5 μL 2×KASP Master Mix (LGC, Lot No. 13426773), and sterile ultrapure water to a final volume of 3 μL.

[0092] The PCR reaction procedure is as follows: Step 1: Pre-denaturation at 94℃ for 15 min;

[0093] Step 2: Denaturation at 94℃ for 20 seconds, annealing for 20 seconds (the first annealing temperature is 65℃, and the temperature is reduced by 1℃ for each cycle) for a total of 10 cycles; denaturation at 94℃ for 20 seconds, annealing at 55℃ for 1 minute for a total of 32 cycles;

[0094] Step 3: Extend at 72℃ for 10 minutes, then store at 4℃.

[0095] The experiment also included a blank control (CK) in the reaction system without template DNA, with two controls per plate.

[0096] 2. Genotyping

[0097] After the PCR reaction, a fluorescence signal reader (Omega) and a fluorescence detection system (Araya) were used to convert the fluorescence signals into analyzable values ​​for fluorescence data reading of the reaction products. The fluorescence scanning results were graphically displayed using the R software package. C-base types (QGPC.caas-7ALa) exhibited FAM fluorescence, distributed near the x-axis; T-base types (QGPC.caas-7ALb) exhibited HEX fluorescence, distributed near the y-axis; samples with no detected signal were distributed near the origin. The FAM excitation wavelength was 485 nm, and the emission wavelength was 520 nm. The HEX excitation wavelength was 535 nm, and the emission wavelength was 556 nm. The system reference fluorescence ROX had an excitation wavelength of 575 nm and an emission wavelength of 610 nm.

[0098] The results are as follows Figure 2As shown, if only the FAM group shows a fluorescent signal, then the genotype of the wheat AX-109534708 to be tested is CC (i.e., the SNP site AX-109534708 in the wheat genome is homozygous for C); if only the HEX group shows a fluorescent signal, then the genotype of the wheat AX-109534708 to be tested is TT (i.e., the SNP site AX-109534708 in the wheat genome is homozygous for T).

[0099] Example 2: Application of the molecular marker KaspAX-109534708 related to grain protein content and its identification primer set. The experimental materials to be tested were 163 wheat varieties, as detailed in Table 4.

[0100] 1. All experimental materials were planted in Anyang, Henan Province in 2012-2013 and 2013-2014. Each material was planted in 4 rows with a row length of 1.5m. Routine management was implemented. After maturity, the grains were harvested, and the grain protein content was determined using a near-infrared spectroscopy analyzer (DA 7250, Perten). The specific detection method followed the national standard GB / T 5506.4-2008. Protein content (14% wet basis) = Protein (dry basis) content × (1-14%). Specific grain protein contents are shown in Table 4.

[0101] 2. The genotype of the wheat SNP locus AX-109534708 was detected using the KaspAX-109534708 marker. The specific experimental steps are as described in step four of Example 1. The genotype analysis results are shown in Table 4.

[0102] Table 4. Genotyping results and grain protein content of 163 wheat varieties.

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Table 5. Statistical analysis results of gene type and grain protein content of wheat SNP locus AX-109534708

[0110]

[0111] Note: Statistical analysis was performed using a two-tailed t-test (P<0.05 indicates that the difference is statistically significant).

[0112] The results are shown in Tables 4 and 5. Figure 2 . Figure 2 In the text, TT indicates that the genotype of the SNP locus AX-109534708 in wheat material is TT, CC indicates that the genotype of the SNP locus AX-109534708 in wheat material is CC, and CK is a blank control in the reaction system without the addition of template DNA.

[0113] KASP marker detection revealed that among 163 Chinese wheat germplasm accessions, 18 were of the TT allele type (i.e., genotype TT at SNP locus AX-109534708), and 145 were of the CC allele type (genotype CC at SNP locus AX-109534708). Wheat germplasm carrying the QTL QGPC.caas-7AL allele type TT had significantly higher grain protein content in different years (13.60% and 13.36% in two years, respectively) than wheat germplasm carrying the QTL QGPC.caas-7AL allele type CC (13.07% and 12.56% in two years, respectively) (P<0.05) (Tables 4 and 5).

[0114] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of substances for detecting SNP polymorphisms or genotypes in the wheat genome in any of the following: (1) To identify or assist in the identification of wheat grain protein content; (2) Breeding related to wheat grain protein content; (3) Prepare products for identification or auxiliary identification of wheat grain protein content; (4) Prepare breeding products related to wheat grain protein content; The SNP site is a SNP site on wheat chromosome 7A, and its nucleotide type is C or T, which is the 36th nucleotide of sequence 1 in the sequence listing. The genotype of the SNP locus is CC or TT, where CC is the homozygous type of the SNP locus C and TT is the homozygous type of the SNP locus T; the grain protein content of the wheat sample with the genotype TT at the SNP locus is higher or candidate higher than that of the wheat sample with the genotype CC at the SNP locus.

2. The application according to claim 1, characterized in that: The substance is either D1), D2), or D3). D1) The substance is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites; D2) The substance is a PCR reagent containing the primer composition described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

3. The application according to claim 2, characterized in that: The primer composition consists of primer A, primer B and primer C; Primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-41 of sequence 2 in the sequence listing; Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-41 of sequence 3 in the sequence listing. The primer C nucleotide sequence is the single-stranded DNA molecule of sequence 4 in the sequence listing.

4. A method for identifying or assisting in the identification of wheat grain protein content, characterized in that: This includes detecting the genotype of a SNP site in the genome of the wheat to be tested, and identifying or assisting in the identification of wheat grain protein content based on the genotype. The SNP site is a SNP site on wheat chromosome 7A, and its nucleotide type is C or T, which is the 36th nucleotide of sequence 1 in the sequence listing. The genotype of the SNP site is CC or TT, where CC is the homozygous type of the SNP site with the genotype C, and TT is the homozygous type of the SNP site with the genotype T. The grain protein content of the wheat to be tested with the genotype TT of the SNP site is higher or is a candidate higher than that of the wheat to be tested with the genotype CC of the SNP site.

5. A method for breeding wheat grains related to protein content, characterized in that: The method includes detecting the genotype of the SNP locus in claim 1 in the wheat genome, selecting wheat with the genotype TT at the SNP locus as a parent for breeding, wherein TT is a homozygous type of the SNP locus T; wherein the genotype of the SNP locus is CC or TT, wherein CC is a homozygous type of the SNP locus C, and TT is a homozygous type of the SNP locus T; and wherein the grain protein content of the wheat to be tested with the genotype TT at the SNP locus is higher than or candidate to be higher than that of the wheat to be tested with the genotype CC at the SNP locus.

6. The application of the method of claim 4 or 5 in breeding related to wheat grain protein content.

7. The product, characterized in that: The product contains the substance of claim 1, and the product is any one of the following: C1) Products that detect single nucleotide polymorphisms or genotypes related to wheat grain protein content; C2) Products used for identifying or assisting in the identification of wheat grain protein content; C3) Products used in breeding programs related to wheat grain protein content; The substance is either D1), D2), or D3). D1) The substance is a primer composition for amplifying wheat genomic DNA fragments including the SNP sites; D2) The substance is a PCR reagent containing the primer composition described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2); The primer composition consists of primer A, primer B and primer C; Primer A is a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing or whose nucleotide sequence is the single-stranded DNA at positions 22-41 of sequence 2 in the sequence listing; Primer B is a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing or whose nucleotide sequence is single-stranded DNA at positions 22-41 of sequence 3 in the sequence listing. The primer C nucleotide sequence is the single-stranded DNA molecule of sequence 4 in the sequence listing.

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