KASP marker for identifying protein content of wheat grains and application of KASP marker

Abstract

The invention discloses a KASP marker for identifying the protein content of wheat grains and application of the KASP marker. The KASP-labeled primer group for identifying the protein content of the wheat grains consists of an upstream primer F1 as shown in SEQ ID NO: 1, an upstream primer F2 as shown in SEQ ID NO: 2 and a downstream primer R as shown in SEQ ID NO: 3. Experiments prove that the primer group provided by the invention can detect the genotype of to-be-detected wheat based on the T100A SNP site, and the T100A SNP site is the 100th nucleotide from the 5'terminal of SEQ ID NO: 4 in a wheat genome, so that the protein content character of wheat grains is quickly and efficiently identified, and the breeding process of a new variety of high-quality wheat is accelerated. The molecular marker has important theoretical significance and economic value for molecular marker-assisted selection of wheat germplasm or breeding progeny materials with high grain protein content.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a KASP marker for identifying the protein content of wheat grains and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is the world's largest cultivated grain with the widest planting area, the widest application range and the highest yield. China is the largest wheat producer and consumer. Wheat is of great significance to ensuring my country's food security. Before the 1980s, my country's wheat breeding work focused on yield, and the genetic improvement of wheat quality started late, and the comprehensive processing quality was lower than that of foreign wheat. In the past 20 years, with the development of society and the improvement of people's living standards, people's demand for high-quality wheat has become more urgent, and wheat quality improvement has been listed as an important breeding goal. According to my country's current "Wheat Variety Quality Classification Standard (GB / T 17320-2013)", wheat quality indicators mainly include grain hardness, crude protein content, and wheat flour wet gluten content, sedimentation value, water absorption, stability time, maximum tensile resistance, energy, etc.

[0003] The protein content of wheat grains is generally between 13% and 15%, with a range of 8-22%. A large number of studies have shown that the protein content and quality of grains have an important impact on the nutritional quality and processing quality of wheat grains. According to the solubility in different solvents, wheat grain proteins can be divided into glutenin, alcohol-soluble proteins, albumin and globulin. Albumin and globulin are collectively referred to as soluble proteins, which mainly affect the nutritional quality of wheat grains. Alcohol-soluble proteins and glutenin are collectively referred to as insoluble proteins, also known as gluten proteins, which mainly affect the processing quality of wheat grains. Grain protein content is a quantitative trait controlled by multiple genes. At present, some QTLs related to wheat grain protein content have been located, but due to factors such as mapping populations, genetic backgrounds and mapping methods, the results can only reflect the genetic information contained in a specific wheat variety, and most QTLs have a small contribution rate to the phenotype and poor repeatability between different environments, so they still cannot meet the needs of molecular marker-assisted selection (MAS).

[0004] Competitive allele-specific PCR (Kompetitive Allele-Specific PCR, KASP) is a fluorescence-based homogeneous genotyping technology developed in recent years. Primers are designed according to specific SNPs or InDels in the target allele, and different fluorescent groups are added to the ends of the primers. Finally, the target sequence is typed based on the reading of the PCR terminal fluorescent signal. It has the advantages of high efficiency, accuracy, and low cost, greatly accelerating the process of molecular marker-assisted selection, and has broad application prospects in crop breeding. Therefore, the development of KASP markers for identifying the protein content of wheat grains can provide an effective detection method for the selection and breeding of new high-quality wheat varieties, which is of great significance to improving the level of high-quality wheat breeding in my country. Summary of the invention

[0005] The purpose of the present invention is to screen or assist in screening wheat with different grain protein contents.

[0006] The present invention firstly protects a method for screening or assisting in screening wheat with different grain protein content, which may include the following steps: detecting whether the genotype of the wheat to be tested is the genotype qGPC6A.1a or the genotype qGPC6A.1b, and the grain protein content of the wheat with the genotype qGPC6A.1b is greater than the grain protein content of the wheat with the genotype qGPC6A.1a;

[0007] The wheat of the genotype qGPC6A.1b is a wheat of the genotype of TT homozygous based on the T100A SNP site;

[0008] The wheat of the genotype qGPC6A.1a is wheat of homozygous AA genotype based on the T100A SNP site;

[0009] The T100A SNP site is the 100th nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0010] In the above method, the step of detecting whether the genotype of the wheat to be tested is the genotype qGPC6A.1a or the genotype qGPC6A.1b may be as follows:

[0011] (a1) using the genomic DNA of the wheat to be tested as a template and using primer set 1 to perform PCR amplification to obtain a PCR amplification product;

[0012] The primer set 1 consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3;

[0013] (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.

[0014] In the above method, the reaction procedure for PCR amplification using primer set 1 can be specifically as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension for 20 s (the first annealing / extension temperature is 61°C, and it decreases by 0.6°C each cycle), for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 1 min, for a total of 26 cycles; extension at 72°C for 3 min; and storage at 4°C.

[0015] In the above method, the reaction system for PCR amplification using primer set 1 can be specifically 1.6 μL, consisting of 0.4 μL 2×KASP Master Mix (LGC, Lot No. 13426773), 0.022 μL primer working solution, 0.8 μL template and ultrapure water. The primer working solution is a mixture of 12 volumes of upstream primer F1 aqueous solution (concentration 100 μM), 12 volumes of upstream primer F2 aqueous solution (concentration 100 μM), 30 volumes of downstream primer R aqueous solution (concentration 100 μM) and 46 volumes of 10 mM Tris-HCl (pH 8.0).

[0016] In the above method, the step of detecting whether the genotype of the wheat to be tested is the genotype qGPC6A.1a or the genotype qGPC6A.1b may be as follows:

[0017] (b1) using the genomic DNA of the wheat to be tested as a template and using primer set 2 to perform PCR amplification to obtain a PCR amplification product;

[0018] The primer set 2 consists of an upstream primer F1 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 1, an upstream primer F2 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3;

[0019] (b2) taking the PCR amplification product obtained in step (b1) and sequencing it;

[0020] (b3) Obtaining the genotype of the wheat to be tested according to the sequencing results obtained in step (b2).

[0021] The present invention also protects a kit for identifying or assisting in identifying the protein content of wheat grains. The kit may include a substance for detecting whether the genotype of the wheat to be tested is the genotype qGPC6A.1a or the genotype qGPC6A.1b;

[0022] The genotype qGPC6A.1b is based on the genotype of the T100A SNP site, which is TT homozygous;

[0023] The genotype qGPC6A.1a is based on the genotype of the T100A SNP site, which is AA homozygous;

[0024] The T100A SNP site is the 100th nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0025] The kit may specifically be composed of a substance for detecting whether the genotype of the wheat to be tested is the genotype qGPC6A.1a or the genotype qGPC6A.1b.

[0026] Any of the above-mentioned substances for detecting whether the genotype of the wheat to be tested is genotype qGPC6A.1a or genotype qGPC6A.1b can specifically be primer set 1; the primer set 1 consists of upstream primer F1 shown in SEQ ID NO: 1, upstream primer F2 shown in SEQ ID NO: 2 and downstream primer R shown in SEQ ID NO: 3.

[0027] Any of the above-mentioned substances for detecting whether the genotype of the wheat to be tested is the genotype qGPC6A.1a or the genotype qGPC6A.1b can specifically be primer set 2; the primer set 2 is composed of the upstream primer F1 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 1, the upstream primer F2 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3.

[0028] The preparation method of the kit also belongs to the protection scope of the present invention. The preparation method of the kit comprises the steps of packaging each primer in any one of the primer sets 1 or any one of the primer sets 2 separately.

[0029] The present invention also protects the molecular marker represented by SEQ ID NO: 4 or the 73rd to 182nd positions from the 5' end of SEQ ID NO: 4.

[0030] The present invention also protects the use of any of the above-mentioned kits or any of the above-mentioned molecular markers in identifying or assisting in identifying the protein content of wheat grains.

[0031] The present invention also protects the use of any of the above-mentioned kits or any of the above-mentioned molecular markers in screening or auxiliary screening of wheat with different grain protein content.

[0032] The present invention also protects the use of any of the above-mentioned kits or any of the above-mentioned molecular markers in wheat breeding.

[0033] In the above, when the genotype of the T100A SNP site in the molecular marker is TT homozygous, the wheat is determined to be of genotype qGPC6A.1b. When the genotype of the T100A SNP site in the molecular marker is AA homozygous, the wheat is determined to be of genotype qGPC6A.1a. The T100A SNP site is the 100th nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0034] The grain protein content of wheat with genotype qGPC6A.1b is greater than that of wheat with genotype qGPC6A.1a.

[0035] In the above, the > may specifically be a statistical >.

[0036] In the above text, the nucleotide sequence shown in the 1st to 21st positions from the 5' end of SEQ ID NO: 1 is a fluorescent tag sequence (i.e., a FAM fluorescent tag sequence), and the fluorescent signal is specifically green. The nucleotide sequence shown in the 1st to 21st positions from the 5' end of SEQ ID NO: 2 is also a fluorescent tag sequence (i.e., a HEX fluorescent tag sequence), and the fluorescent signal is specifically pink.

[0037] Experiments have shown that the method provided by the present invention can be used to detect the genotype of the wheat to be tested based on the T100A SNP site, and can screen or assist in screening the protein content trait of wheat grains. At the same time, the method provided by the present invention also has the advantages of high throughput, accuracy, low cost, simple operation, saving manpower and material resources, etc. The present invention can accelerate the breeding process of new high-quality wheat varieties and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The T100A SNP site and the positions of three primers on the DNA molecule shown in SEQ ID NO:4.

[0039] Figure 2 The results of genotyping of 315 wheat germplasms in natural populations (CK is the negative control; AA is the AA homozygous type, i.e., genotype qGPC6A.1a; TT is the TT homozygous type, i.e., genotype qGPC6A.1b).

[0040] Figure 3This is an association analysis between wheat germplasm of genotype qGPC6A.1b and genotype qGPC6A.1a and the mean value of grain protein content in natural populations under different environments; among them, 20-21normal_A is the genotype qGPC6A.1a of normal water and fertilizer in 2020-2021, 20-21normal_T is the genotype qGPC6A.1b of normal water and fertilizer in 2020-2021, 21-22normal_A is the genotype qGPC6A.1a of normal water and fertilizer in 2021-2022, 21-22normal_T is the genotype qGPC6A.1b of normal water and fertilizer in 2021-2022, 20-21drought_A is the genotype qGPC6A.1a of drought treatment in 2020-2021, and 20-21drought_T is the genotype qGPC6A.1b of drought treatment in 2020-2021. GPC6A.1b, 21-22 drought_A is the genotype qGPC6A.1a of the drought treatment in 2021-2022, 21-22 drought_T is the genotype qGPC6A.1b of the drought treatment in 2021-2022, 20-21 low nitrogen_A is the genotype qGPC6A.1a of the low nitrogen treatment in 2020-2021, 20-21 low nitrogen_T is the genotype qGPC6A.1b of the low nitrogen treatment in 2020-2021, 21-22 low nitrogen_A is the genotype qGPC6A.1a of the low nitrogen treatment in 2021-2022, 21-22 low nitrogen_T is the genotype qGPC6A.1b of the low nitrogen treatment in 2021-2022, ** represents P < 0.01, that is, the difference reaches an extremely significant level; * represents P < 0.05, that is, the difference reaches a significant level. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0042] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0043] The wheat materials used in the following examples are all from the Wheat Research Center of the Institute of Cereals and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences. The public can obtain the biological materials from the applicant. The biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes. Since the wheat materials are all cultivated varieties, they are usually assumed to be highly homozygous plant materials, and the genotypes are all homozygous.

[0044] Example 1. Discovery of the T100A SNP site in the wheat genome and establishment of a wheat genotyping method based on the T100A SNP site

[0045] 1. Discovery of the T100A SNP site in the wheat genome

[0046] After a large number of experiments, the inventors of the present invention found a SNP site in the wheat genome, named T100ASNP site. The T100A SNP site is located at the 100th position from the 5' end of SEQ ID NO: 4, and the genotypes are TT homozygous and AA homozygous. In the Chinese spring wheat genome IWGSC RefSeq v1.0, the physical position of the T100A SNP site is the 597992358th position of chromosome 6A; the physical position of the DNA molecule shown in SEQ ID NO: 4 is the 597992259-597992458th position of chromosome 6A.

[0047] 2. Synthesis of primer sets for amplification of T100A SNP loci

[0048] Based on the KASP technical principle, the inventors of the present invention designed and synthesized a primer set for amplifying the T100ASNP site after a large number of experimental explorations. The primer set consists of an upstream primer F1, an upstream primer F2 and a downstream primer R. The nucleotide sequences of each primer are shown in Table 1.

[0049] Table 1

[0050]

[0051] Note: Single underline is FAM fluorescent tag sequence, double underline is HEX fluorescent tag sequence.

[0052] The positions of the T100A SNP site and the three primers (parts) on the DNA molecule shown in SEQ ID NO: 4 are shown in FIG. Figure 1 (The red background represents the T100A SNP site, and the boxes indicate primers).

[0053] 3. Establishment of the genotyping method based on T100A SNP locus for wheat to be tested

[0054] After a large number of experiments, the inventors of the present invention established a genotyping method for wheat to be tested based on the T100A SNP site. The specific steps are as follows:

[0055] 1. Obtaining genomic DNA of wheat to be tested

[0056] The genomic DNA of the young leaves of the wheat to be tested was extracted by the CTAB method to obtain the genomic DNA of the wheat to be tested.

[0057] The quality and concentration of the genomic DNA of the wheat to be tested must meet the PCR requirements. The standards are: 1% agarose electrophoresis shows clear DNA bands, no obvious impurities, and no degradation; the UV spectrophotometer Nanodrop2000 (Thermo) detects an A260 / A280 ratio of about 1.8 and an A260 / A230 ratio greater than 1.8.

[0058] The genomic DNA of the wheat to be tested was diluted with deionized water to a concentration of 28.3 ng / μL to obtain a template for PCR amplification in the next step.

[0059] 2. Using the genomic DNA of the wheat to be tested or water (as a negative control) as a template, perform PCR amplification using the primer set synthesized in step 2 to obtain a PCR amplification product.

[0060] The reaction system was 1.6 μL, consisting of 0.4 μL 2×KASP Master Mix (LGC, Lot No. 13426773), 0.022 μL primer working solution, 0.8 μL template and ultrapure water. The primer working solution was a mixture of 12 μL upstream primer F1 aqueous solution (100 μM concentration), 12 μL upstream primer F2 aqueous solution (100 μM concentration), 30 μL downstream primer R aqueous solution (100 μM concentration) and 46 μL 10 mM Tris-HCl (pH 8.0).

[0061] The reaction procedure was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, renaturation / extension for 20 s (the first renaturation / extension temperature was 61°C, and decreased by 0.6°C for each cycle), for a total of 10 cycles; denaturation at 94°C for 20 s, renaturation / extension at 55°C for 1 min, for a total of 26 cycles; extension at 72°C for 3 min; and storage at 4°C.

[0062] 3. After completing step 2, the fluorescence data of the PCR amplification product was read using a fluorescence detection system (Araya), and the results were graphically displayed using GraphPad Prism, that is, the genotype of the wheat to be tested was determined based on the color of the fluorescence signal. The specific principles are as follows: If the wheat to be tested shows a green fluorescence signal, the genotype of the wheat to be tested based on the T100A SNP site is AA homozygous, and the genotype of the wheat to be tested is named genotype qGPC6A.1a; if the wheat to be tested shows a pink fluorescence signal, the genotype of the wheat to be tested based on the T100A SNP site is TT homozygous, and the genotype of the wheat to be tested is named genotype qGPC6A.1b. The negative control has no detection signal.

[0063] The graphical display results are as follows: the genotype qGPC6A.1b showing a pink fluorescent signal is distributed near the y-axis; the genotype qGPC6A.1a showing a green fluorescent signal is distributed near the x-axis; and the negative control with no detected signal is distributed near the origin.

[0064] Example 2. Association analysis between two wheat genotypes (genotype qGPC6A.1b and genotype qGPC6A.1a) and grain protein content

[0065] 1. Genotyping of wheat in natural populations

[0066] The method of step 3 in Example 1 was used to perform genotyping on each wheat variety in the natural population. The natural population consisted of 315 wheat germplasms (all hexaploid) representing the main varieties and excellent lines in the Huanghuai wheat region. The names of the wheat germplasms are detailed in the second column of Table 2.

[0067] Test results see Figure 2 The genotypes of the 315 wheat accessions are detailed in column 3 of Table 2. The genotypes of 158 wheat accessions were genotype qGPC6A.1a, and the genotypes of 157 wheat accessions were genotype qGPC6A.1b.

[0068] Table 2

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Note: NA indicates that the data on average grain protein content are missing.

[0079] 2. Detection of wheat grain protein content traits

[0080] 315 wheat germplasms from natural populations were planted at the Di Shang Experimental Station of the Institute of Cereals and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences for two consecutive years (2020-2021 and 2021-2022). They were sown every year under normal water and fertilizer conditions (watered once at the jointing stage and once at the filling stage, with an irrigation volume of 50m 3 / mu, nitrogen fertilizer was 12Kg / mu), drought treatment (no watering during the whole growth period, nitrogen fertilizer was 12Kg / mu) and low nitrogen treatment (watering once during the jointing stage and the filling stage, with an irrigation volume of 50m 3 / mu, nitrogen fertilizer 6 kg / mu) 3 environments, 3 m row length, randomized block design, three replicates. After harvest, 30 grains of each wheat germplasm were randomly selected, and the protein content of wheat grains was determined using a DA7200 near-infrared analyzer (Perten) and the average was taken, recorded as the average grain protein content.

[0081] The statistical results of the average grain protein content of 315 wheat germplasms under six environments (i.e., normal water and fertilizer in 2020-2021, normal water and fertilizer in 2021-2022, drought treatment in 2020-2021, drought treatment in 2021-2022, low nitrogen treatment in 2020-2021, and low nitrogen treatment in 2021-2022) are shown in columns 4-9 in Table 2. Only some wheat germplasms did not obtain the average grain protein content data due to planting reasons, which are indicated by NA.

[0082] 3. Correlation Analysis

[0083] 1. Take the average grain protein content of wheat germplasm of the same genotype under the same environment and record it as the mean grain protein content.

[0084] 2. The two-tailed t-test was used to conduct an association analysis between the wheat germplasm of genotype qGPC6A.1b and genotype qGPC6A.1a and the mean value of grain protein content in natural populations under the same environment.

[0085] The results are shown in Table 3 (the number of wheat germplasms was obtained according to Table 2, i.e., the number of wheat germplasms for which the average grain protein content data was not obtained was removed) and Figure 3 .

[0086] Table 3

[0087]

[0088] Note: P<0.05 means the difference is significant; P<0.01 means the difference is extremely significant.

[0089] The results showed that in a natural population of 315 wheat germplasms, the grain protein content of wheat with genotype qGPC6A.1b was greater than that of wheat with genotype qGPC6A.1a, and the ">" was statistically greater. Studies on natural populations have shown that genotype qGPC6A.1b is an excellent genotype for increasing the protein content of wheat grains.

[0090] The above results show that by detecting the genotype of the wheat to be tested based on the T100A SNP site, the wheat grain protein content trait can be screened or assisted in screening, which has important application value in the process of wheat molecular marker-assisted breeding.

[0091] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for screening or assisting in screening wheat with different grain protein content, comprising the following steps: detecting whether the genotype of the wheat to be tested is genotype qGPC6A.1a or genotype qGPC6A.1b, and the grain protein content of the wheat with genotype qGPC6A.1b is greater than the grain protein content of the wheat with genotype qGPC6A.1a; The wheat of the genotype qGPC6A.1b is a wheat of the genotype of TT homozygous based on the T100A SNP site; The wheat of the genotype qGPC6A.1a is wheat of homozygous AA genotype based on the T100A SNP site; The T100A SNP site is the 100th nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

2. The method according to claim 1, characterized in that: The steps of detecting whether the genotype of the wheat to be tested is genotype qGPC6A.1a or genotype qGPC6A.1b are as follows: (a1) using the genomic DNA of the wheat to be tested as a template and using primer set 1 to perform PCR amplification to obtain a PCR amplification product; The primer set 1 consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO: 3; (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 genotype of the wheat to be tested is genotype qGPC6A.1a or genotype qGPC6A.1b are as follows: (b1) using the genomic DNA of the wheat to be tested as a template and using primer set 2 to perform PCR amplification to obtain a PCR amplification product; The primer set 2 consists of the upstream primer F1 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 1, the upstream primer F2 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3; (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 protein content of wheat grains, comprising a substance for detecting whether the genotype of the wheat to be tested is genotype qGPC6A.1a or genotype qGPC6A.1b; The genotype qGPC6A.1b is based on the genotype of the T100A SNP site, which is TT homozygous; The genotype qGPC6A.1a is based on the genotype of the T100A SNP site, which is AA homozygous; The T100A SNP site is the 100th nucleotide from the 5' end of SEQ ID NO: 4 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 genotype qGPC6A.1a or genotype qGPC6A.1b is primer set 1; The primer set 1 consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO:

3.

6. The kit according to claim 4, characterized in that: The material for detecting whether the genotype of the wheat to be tested is genotype qGPC6A.1a or genotype qGPC6A.1b is primer set 2; The primer set 2 consists of an upstream primer F1 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 1, an upstream primer F2 shown at positions 22 to 49 from the 5' end of SEQ ID NO: 2, and a downstream primer R shown in SEQ ID NO:

3.

7. The molecular marker represented by SEQ ID NO: 4 or by positions 73 to 182 from the 5' end of SEQ ID NO:

4.

8. Use of the kit according to any one of claims 4 to 6 or the molecular marker according to claim 7 in identifying or assisting in identifying the protein content of wheat grains.

9. Use of the kit according to any one of claims 4 to 6 or the molecular marker according to claim 7 in screening or assisting screening of wheat with different grain protein content.

10. Use of the kit according to any one of claims 4 to 6 or the molecular marker according to claim 7 in wheat breeding.

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