KASP marker for identifying gelatinization characteristics of wheat starch in multiple environments and application of KASP marker

Abstract

The invention discloses a KASP marker for identifying wheat starch gelatinization characteristics in multiple environments and application, and relates to the technical field of biology, in particular to a method for screening or assisting in screening wheat with different peak viscosities, which comprises the following steps: detecting whether the genotype of wheat to be detected is qRVA-Peak7B.1a or qRVA-Peak7B.1b, and the peak viscosity gt of the wheat with the genotype qRVA-Peak7B.1b; the peak viscosity of the wheat of the genotype qRVA-Peak7B. 1a; the wheat of which the genotype is qRVA-Peak7B. 1b is wheat of which the genotype is TT homozygous type based on a C101T SNP (Single Nucleotide Polymorphism) site; the wheat of which the genotype is qRVA-Peak7B.1a is wheat of which the genotype is CC homozygous based on a C101T SNP (Single Nucleotide Polymorphism) site; the C101T SNP locus is the 101 nucleotide from the 5'terminal of SEQ ID NO.4 in a wheat genome. The molecular marker has important theoretical significance and economic value for molecular marker-assisted selection of wheat germplasm or breeding progeny materials with relatively high peak viscosity (important indexes of starch gelatinization characteristics).

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a KASP marker for identifying the gelatinization properties of wheat starch under multiple environments and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is the most widely cultivated and most consumed crop in the world. It is also one of the three major grain crops in my country and is of great strategic significance to ensuring national food security. China's wheat production and consumption are both ranked first in the world. With the development of social economy and the improvement of people's living standards, wheat quality has received great attention from breeders and consumers, and quality improvement has become one of the main tasks of wheat breeding in my country. Starch, as the main component of wheat grains (accounting for about 75% of grains), has an important influence on wheat quality. The gelatinization characteristics of wheat starch are one of the most important indicators for evaluating the quality of wheat starch, and are closely related to the quality of foods such as noodles. Therefore, studying the gelatinization characteristics of wheat starch and its genetic basis is of great significance to wheat quality improvement.

[0003] Evaluation indicators of wheat starch gelatinization properties include peak viscosity, trough viscosity, dilution value, final viscosity, rebound value, peak time and gelatinization temperature. Among them, the most important indicator for measuring starch gelatinization properties is peak viscosity. Generally speaking, noodles made from wheat flour with high peak viscosity have better quality. In addition, studies have shown that there is a significant positive correlation between peak viscosity and the elasticity, smoothness and softness of noodles. Peak year is a quantitative trait inheritance, and its inheritance is controlled by multiple genes, mainly located on chromosomes 1A, 1B, 3A, 3B, 4A, 5A, 5B, 6A, 6B, 7A and 7B. However, due to the influence of factors such as mapping population, genetic background and mapping method, 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] Molecular marker-assisted selection is a modern breeding method that uses DNA molecular markers that are closely linked to the target trait to select the genotype of the target trait. It has the advantage of not being affected by external environmental factors. KASP (Kompetitive Allele-Specific PCR) 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. 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, and has broad application prospects in crop breeding. Therefore, the development of KASP markers for identifying the gelatinization characteristics of wheat starch 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 technical problem to be solved by the present invention is to provide a KASP marker for identifying the gelatinization characteristics of wheat starch under multiple environments and its application.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] A SNP site associated with wheat peak viscosity, the SNP site is located on wheat chromosome 7B, the physical position of the SNP site in the wheat genome version number IWGSC RefSeq v1.0 is 135576110bp, the SNP site corresponds to the 101st base from the 5' end of SEQ ID NO: 4, when the site is TT homozygous, the corresponding genotype is qRVA-Peak7B.1b; when the site is CC homozygous, the corresponding genotype is qRVA-Peak7B.1a; the peak viscosity is: the peak viscosity of wheat with the genotype qRVA-Peak7B.1b is greater than or has a candidate greater than the peak viscosity of wheat with the genotype qRVA-Peak7B.1a.

[0008] A reagent or kit for identifying or assisting in identifying the peak viscosity trait of wheat, the reagent or kit is used to detect the SNP site described in claim 1, and the reagent or kit comprises a PCR amplification specific primer combination corresponding to the SNP site, as well as template DNA, buffer, dNTPs and other necessary components for gene detection.

[0009] Further preferably, the PCR amplification specific primer combination 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.

[0010] A primer combination is used to detect the single nucleotide polymorphism of the following SNP site in the wheat genome, wherein the SNP site is located on the wheat chromosome 7B, the physical position of the SNP site in the wheat genome version number IWGSC RefSeq v1.0 is 135576110bp, the SNP site corresponds to the 101st base from the 5' end of SEQ ID NO: 4, when the site is TT homozygous, the corresponding genotype is qRVA-Peak7B.1b; when the site is CC homozygous, the corresponding genotype is qRVA-Peak7B.1a; the peak viscosity is: the peak viscosity of the wheat with the genotype qRVA-Peak7B.1b is greater than or candidate greater than the peak viscosity of the wheat with the genotype qRVA-Peak7B.1a; the PCR amplification specific primer combination consists of an upstream primer F1 shown in SEQ ID NO: 1, an upstream primer F2 shown in SEQ ID NO: 2 and a primer F3 shown in SEQ ID NO: 3. NO: The downstream primer R composition shown in 3; this primer combination is used to detect the SNP site described in claim 1.

[0011] Application of the above SNP loci in wheat molecular marker-assisted selection breeding.

[0012] The above SNP sites are used for primary screening, rescreening, identification, classification, and / or auxiliary identification of wheat peak viscosity in the early stage of wheat molecular marker-assisted selection breeding.

[0013] The application of the primer combination in the directional breeding or auxiliary directional breeding of wheat lines with high peak viscosity.

[0014] The method for identifying or assisting in identifying the peak viscosity of wheat in the early stage of breeding comprises the following steps:

[0015] (1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification to obtain a PCR amplification product;

[0016] The primer combination 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;

[0017] (2) After completing step (1), 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;

[0018] (3) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than that of wheat with genotype qRVA-Peak7B.1a.

[0019] The method for identifying or assisting in identifying the peak viscosity of wheat in the early stage of breeding comprises the following steps:

[0020] (1) using the genomic DNA of the wheat to be tested as a template and using a primer combination to perform PCR amplification to obtain a PCR amplification product;

[0021] The primer combination 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;

[0022] (2) taking the PCR amplification product obtained in step (1) and sequencing it;

[0023] (3) obtaining the genotype of the wheat to be tested according to the sequencing results obtained in step (2);

[0024] (4) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than that of wheat with genotype qRVA-Peak7B.1a.

[0025] Based on the above SNP sites, KASP molecular markers related to wheat peak viscosity were developed.

[0026] The beneficial effect of adopting the above technical scheme is that: the present invention provides a KASP marker Kasp_qRVA-Peak7B.1 for identifying the allelic variation of qRVA-Peak7B.1a and qRVA-Peak7B.1b and its correlation with the peak viscosity, an important indicator of wheat starch gelatinization characteristics. The SNP molecular marker in the present invention is applied to the molecular marker-assisted selection of wheat starch gelatinization characteristics, which can quickly and efficiently screen out wheat varieties (germplasm) with higher peak viscosity, thereby accelerating the breeding process of high-quality new wheat varieties. The present invention has important theoretical significance and economic value for the use of molecular markers to assist in the selection of wheat germplasm or breeding offspring materials with higher peak viscosity (an important indicator of starch gelatinization characteristics). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the KASP primer positions of different allele types of QTL qRVA-Peak7B.1 related to starch gelatinization characteristics of common wheat on chromosome 7B; the sequence in the figure is the sequence of physical position 135576010bp-135576209bp of wheat chromosome 7B, the upstream and downstream primer positions of the KASP marker are marked with boxes, and the red background represents the SNP at the physical position 135576110bp of chromosome 7B.

[0028] Figure 2Schematic diagram of the detection results of genotyping of 474 wheat germplasms in natural populations; CC is the CC homozygous type, that is, the genotype qRVA-Peak7B.1a; TT is the TT homozygous type, that is, the genotype qRVA-Peak7B.1b; CK is the negative control.

[0029] Figure 3 Schematic diagram of the association analysis results of wheat germplasm with genotype qRVA-Peak7B.1a and genotype qRVA-Peak7B.1b and the mean value of wheat peak viscosity (an important indicator of starch gelatinization characteristics) in natural populations under different environments; among them, 19-20Normal_C is the genotype qRVA-Peak7B.1a with normal water and fertilizer in 2019-2020, and 19-20Normal_T is the genotype qRVA-Peak7B.1a with normal water and fertilizer in 2019-2020. genotype qRVA-Peak7B.1b, 20-21normal_C is the genotype qRVA-Peak7B.1a of normal water and fertilizer in 2020-2021, 20-21normal_T is the genotype qRVA-Peak7B.1b of normal water and fertilizer in 2020-2021, 19-20drought_C is the genotype qRVA-Peak7B.1a of drought treatment in 2019-2020, 19-20drought_T is the genotype qRVA-Peak7B.1a of drought treatment in 2019-2020, 019-2020 drought treatment genotype qRVA-Peak7B.1b, 20-21 drought_C is the genotype qRVA-Peak7B.1a of the 2020-2021 drought treatment, 20-21 drought_T is the genotype qRVA-Peak7B.1b of the 2020-2021 drought treatment, 19-20 low nitrogen_C is the genotype qRVA-Peak7 B.1a, 19-20 low nitrogen_T is the genotype qRVA-Peak7B.1b of the low nitrogen treatment in 2019-2020, 20-21 low nitrogen_C is the genotype qRVA-Peak7B.1a of the low nitrogen treatment in 2020-2021, and 20-21 low nitrogen_T is the genotype qRVA-Peak7B.1b of the low nitrogen treatment in 2020-2021; ** represents P < 0.01, that is, the difference reaches an extremely significant level. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Example 1: Detection of different allele types of wheat starch gelatinization property-related QTL qRVA-Peak7B.1 using KASP marker Kasp_qRVA-Peak7B.1

[0037] The KASP marker Kasp_qRVA-Peak7B.1 was used to detect the QTL qRVA-Peak7B.1 related to the gelatinization characteristics of wheat starch. The different allele types at the physical position 135576110bp of chromosome 7B (refer to the Chinese spring wheat genome IWGSC RefSeq v1.0) were divided into the following two steps: PCR amplification and genotyping. The experimental methods in the embodiments are all conventional methods unless otherwise specified. Common wheat germplasm materials are all preserved in the Wheat Research Center of the Institute of Cereals and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences.

[0038] (1) PCR amplification system and procedure

[0039] Extract common wheat genomic DNA using the CTAB method and dissolve it in 300 μL ultrapure water. Detect DNA quality by 1% agarose gel electrophoresis, requiring clear bands, no obvious impurities, and no degradation. Measure the concentration and dilute the DNA to 28.3 ng / μL. Use the diluted genomic DNA as a template for PCR amplification.

[0040] Preparation of KASP marker primer working solution: Two KASP upstream primers were designed based on the SNP at the physical position of 135576110bp of wheat starch gelatinization QTL qRVA-Peak7B.1 in the wheat genome version number IWGSC RefSeq v1.0. The polymorphism of the SNP site is a C / T base difference. FAM and HEX fluorescent linker sequences were added to the 5' end of the primers, respectively. At the same time, a universal KASP downstream primer was designed. The primer sequences are shown in Table 1. The KASP marker primer working solution includes: 12 μL of each of the two upstream primers (100 μM), 30 μL of the downstream primer (100 μM), and 46 μL of ultrapure water. After mixing, store at -20°C for later use.

[0041] Table 1 Sequence list of KASP marker primers used to identify allelic variation of QTLqRVA-Peak7B.1 in common wheat

[0042]

[0043]

[0044] PCR amplification system: template DNA 1.5 μL, primer working solution 0.0417 μL, 2×KASP Master Mix (LGC Company, Lot No. 13426773) 0.75 μL, and sterile ultrapure water to supplement the reaction system to 3 μL.

[0045] PCR reaction program: 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 was 61°C, and decreased by 0.6°C for each cycle), 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 1 min, 26 cycles; extension at 72°C for 3 min; storage at 4°C.

[0046] (2) Genotyping

[0047] After the PCR reaction was completed, the fluorescence signal was converted into an analyzable value using a fluorescence signal reader (Omega) and a fluorescence detection system (Araya). The fluorescence scanning results were graphically displayed using GraphPad Prism. qRVA-Peak7B.1a (CC homozygous) had FAM fluorescence, which was distributed near the x-axis; qRVA-Peak7B.1b (TT homozygous) had HEX fluorescence, which was distributed near the y-axis; the negative control (CK) had no detected signal, which was distributed near the origin (e.g. Figure 2 ).

[0048] Example 2: Detection of starch gelatinization characteristics of common wheat germplasm materials using KASP marker Kasp_qRVA-Peak7B.1

[0049] 474 Chinese wheat germplasm materials 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 (2019-2020 and 2020-2021). They were sown every year under normal water and fertilizer conditions (one watering each at the jointing stage and the filling stage, with an irrigation volume of 50m 3 / mu, nitrogen fertilizer 12Kg / mu), drought treatment (no watering during the whole growth period, nitrogen fertilizer 12Kg / mu) and low nitrogen treatment (watering once at the jointing stage and once at the filling stage, with an irrigation volume of 50m 3 / mu, nitrogen fertilizer 6Kg / mu) under 3 environments, 3m row length, randomized block design, and three replications.

[0050] After harvest, the peak viscosity, an important indicator of wheat starch gelatinization characteristics, was measured using a Perten rapid viscometer (RVA). According to KASP marker detection, among the 474 Chinese wheat germplasm materials, 20 germplasms were of the qRVA-Peak7B.1a allele type and 454 germplasms were of the qRVA-Peak7B.1b allele type. The allele types of wheat germplasm materials and the peak viscosity of wheat (an important indicator of starch gelatinization characteristics) in different years and environments are shown in Table 2. The statistical results showed that the mean peak viscosity of wheat germplasm materials carrying the allele qRVA-Peak7B.1b in different years and environments was higher than the mean peak viscosity of wheat germplasm materials carrying the allele qRVA-Peak7B.1a, and the two were extremely significantly different (P<0.01). Table 2 Wheat germplasm Kasp_qRVA-Peak7B.1 marker detection results and peak viscosity of important indicators of wheat starch gelatinization characteristics in different years and environments

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] Note: NA indicates average peak viscosity data are missing.

[0070] The statistical results showed that the mean peak viscosity of wheat germplasm materials carrying the allele qRVA-Peak7B.1b in different years and under different environments was higher than that of wheat germplasm materials carrying the allele qRVA-Peak7B.1a, and the difference between the two was extremely significant (P<0.01) (Table 3, Figure 3 ).

[0071] Table 3 Statistical analysis of the relationship between allelic variation types of QTLqRVA-Peak7B.1 in common wheat and peak viscosity, an important index of wheat starch gelatinization characteristics

[0072]

[0073] Note: P<0.01 means the difference is extremely significant.

[0074] After a large number of experiments, the inventors of the present invention found a SNP site in the wheat genome, named C101T SNP site. The C101T SNP site is located at the 101st position from the 5' end of SEQ ID NO: 4, and the genotype is CC homozygous and TT homozygous. In the Chinese spring wheat genome IWGSC RefSeq v1.0, the physical position of the C101T SNP is the 135576110th position of chromosome 7B. According to the SNP (C / T) difference site, a KASP marker Kasp_qRVA-Peak7B.1 for identifying the gelatinization properties of wheat starch was developed. The allele type qRVA-Peak7B.1a (CC homozygous) carrying FAM fluorescence and distributed near the x-axis is to reduce the peak viscosity of wheat (an important indicator of starch gelatinization properties), and the allele type qRVA-Peak7B.1b (TT homozygous) carrying HEX fluorescence and distributed near the y-axis is to increase the peak viscosity of wheat (an important indicator of starch gelatinization properties). The marker was verified using 474 Chinese wheat germplasm materials under three environments of normal water and fertilizer, drought treatment and low nitrogen treatment for two consecutive years. The results showed that the marker can effectively type the two allele types of qRVA-Peak7B.1a and qRVA-Peak7B.1b. The present invention provides an effective detection method for accurately and quickly screening wheat germplasm materials with high peak viscosity (an important indicator of starch gelatinization characteristics) under multiple environments.

[0075] 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.

[0076] 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.

[0077] 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. A SNP site associated with wheat peak viscosity, characterized in that: The SNP site is located on wheat chromosome 7B, and the physical position of the SNP site in the wheat genome version number IWGSC RefSeq v1.0 is 135576110 bp. The SNP site corresponds to the 101st base from the 5' end of SEQ ID NO:

4. When the site is homozygous for TT, the corresponding genotype is qRVA-Peak7B.1b; when the site is homozygous for CC, the corresponding genotype is qRVA-Peak7B.1a; the peak viscosity is: the peak viscosity of wheat with the genotype qRVA-Peak7B.1b is greater than or has a candidate greater than the peak viscosity of wheat with the genotype qRVA-Peak7B.1a.

2. A reagent or kit for identifying or assisting in identifying the peak viscosity trait of wheat, characterized in that: The reagent or kit is used to detect the SNP site described in claim 1, and the reagent or kit comprises a PCR amplification specific primer combination corresponding to the SNP site, as well as template DNA, buffer, dNTPs and other necessary components for gene detection.

3. The reagent or kit according to claim 2, characterized in that: The PCR amplification specific primer combination 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.

4. A primer combination, characterized in that: Used to detect the single nucleotide polymorphism of the following SNP site in the wheat genome, the SNP site is located on the wheat chromosome 7B, the physical position of the SNP site in the wheat genome version number IWGSCRefSeq v1.0 is 135576110 bp, the SNP site corresponds to the 101st base from the 5' end of SEQ ID NO: 4, when the site is TT homozygous, the corresponding genotype is qRVA-Peak7B.1b; when the site is CC homozygous, the corresponding genotype is qRVA-Peak7B.1a; the peak viscosity is: the peak viscosity of wheat with the genotype qRVA-Peak7B.1b is greater than or candidate greater than the peak viscosity of wheat with the genotype qRVA-Peak7B.1a; the PCR amplification specific primer combination consists of the upstream primer F1 shown in SEQ ID NO: 1, the upstream primer F2 shown in SEQ ID NO: 2 and the downstream primer R shown in SEQ ID NO: 3; this primer combination is used to detect the SNP site described in claim 1.

5. Use of the SNP locus according to claim 1 in wheat molecular marker-assisted selection breeding.

6. Use of the SNP locus described in claim 1 in the initial screening, and / or rescreening, and / or identification, and / or classification, and / or auxiliary identification of wheat peak viscosity in the early stage of wheat molecular marker-assisted selection breeding.

7. Use of the primer combination according to claim 4 in directed breeding or assisted directed breeding of wheat lines with high peak viscosity.

8. A method for identifying or assisting in identifying wheat peak viscosity in the early stages of breeding, characterized in that: The following steps are involved: (1) Using the genomic DNA of the wheat to be tested as a template, a primer combination is used for PCR amplification to obtain a PCR amplification product; The primer combination 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; (2) After completing step (1), an instrument is used to detect the fluorescence signal of the PCR amplification product, and the genotype of the wheat to be tested is obtained according to the color of the fluorescence signal; (3) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than the peak viscosity of wheat with genotype qRVA-Peak7B.1a.

9. A method for identifying or assisting in identifying wheat peak viscosity in the early stages of breeding, characterized in that: The following steps are involved: (1) Using the genomic DNA of the wheat to be tested as a template, a primer combination is used for PCR amplification to obtain a PCR amplification product; The primer combination 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; (2) taking the PCR amplification product obtained in step (1) and sequencing it; (3) Obtaining the genotype of the wheat to be tested according to the sequencing results obtained in step (2); (4) The peak viscosity of wheat with genotype qRVA-Peak7B.1b is greater than the peak viscosity of wheat with genotype qRVA-Peak7B.1a.

10. According to claim 1, the SNP locus is used to develop a KASP molecular marker associated with wheat peak viscosity.

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