Primer combination for screening wheat precipitation value and application thereof

Abstract

The invention discloses a primer combination for screening wheat precipitation value and application of the primer combination. The primer combination for screening the wheat precipitation value 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 combination provided by the invention can detect the genotype of to-be-detected wheat based on the C101GSNP site, the C101G SNP site is the 101th nucleotide from the 5'terminal of SEQ ID NO: 4 in a wheat genome, and then the wheat precipitation value character is rapidly and efficiently identified, so that 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 precipitation value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a primer combination for screening wheat precipitation value and application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is the world's largest cultivated grain with the widest range of applications and the highest yield. Wheat is rich in protein, starch, etc. It can be processed into a variety of foods, providing the main nutrients for people's daily diet. The quality of its final product mainly depends on the quality of flour. Therefore, when breeders, flour mills and food processing companies select high-quality wheat varieties, a basic task is to evaluate the quality of flour, especially in the early selection of quality improvement breeding, thousands of hybrid offspring are identified every year. In order to maximize the efficiency of breeding work, measurement methods that require a small amount of samples, are easy and quick to operate, and have strong repeatability are becoming more and more popular.

[0003] The level of wheat production and quality are directly related to human food satisfaction and nutritional balance. Wheat has unique processing qualities. Wheat flour for different purposes has different requirements for wheat protein content. High-quality strong-gluten wheat is an important raw material for making high-quality bread, noodles, dumplings and other foods. With the rapid development of my country's economy and the improvement of people's living standards, the pasta consumption market is constantly upgrading, and the demand for high-quality strong-gluten wheat is increasing. Therefore, the development of high-quality strong-gluten wheat has become an important goal of wheat breeding in my country and the main direction of industrial structure adjustment.

[0004] Wheat processing quality is divided into two aspects: the first aspect is the milling quality (i.e. primary processing quality), with grain bulk density, hardness index and flour yield as the main evaluation indicators; the second aspect is the food processing quality (i.e. secondary processing quality), with flour gluten content and quality, and dough rheological properties as the main evaluation indicators. Flour and dough quality directly affect the processing quality of foods such as bread, noodles and dumplings. Scientific evaluation of wheat varieties requires a comprehensive analysis of grain quality, flour quality and dough quality. Among them, quality indicators such as sedimentation value, hardness index, gluten index, and dough rheological properties are mainly affected by genotype and are often used as important parameters to analyze and evaluate wheat quality.

[0005] Sedimentation value is extremely important in wheat processing quality indicators. It is a comprehensive indicator for measuring gluten quality and quantity. It is closely related to wheat bread baking quality, dough characteristics and other indicators, and has a high heritability, so it is often used to screen low-generation materials in wheat quality breeding. Studies have shown that sedimentation value is significantly or extremely significantly positively correlated with most flour map parameters (such as dough formation time, stability time, etc.), tensile map parameters (such as tensile area, tensile strength, etc.) and bread scores, and is also significantly positively correlated with protein content and wet gluten content. The larger the sedimentation value of the wheat variety, the greater the gluten strength and the better the baking quality of the flour. However, since the sedimentation value determination is carried out using wheat flour, the wheat sample must have a certain amount, and it is also related to the after-ripening of flour after milling, so it must be placed for one week after milling before testing. Therefore, the application of sedimentation value detection in wheat quality evaluation and wheat breeding is greatly limited. At the same time, wheat processing quality is a relatively complex quantitative trait, which is easily affected by factors such as genetic characteristics, ecological environment and cultivation management, and it is inconvenient to give it a comprehensive evaluation.

[0006] Competitive allele-specific PCR (Kompetitive Allele-Specific PCR, KASP) is a fluorescence-based homogeneous genotyping technology. Primers are designed according to the specific SNPs or InDels contained in the target allele, and different fluorescent groups are added to the ends of the primers. Finally, the target sequence is genotyped based on the reading of the PCR terminal fluorescent signal. It has the advantages of high efficiency, low cost, fast and convenient, greatly accelerating the process of molecular marker-assisted selection, and has broad application prospects in crop breeding. Summary of the invention

[0007] The purpose of the present invention is to screen or assist in screening wheat with different precipitation values.

[0008] The present invention firstly protects a method for screening or assisting screening of wheat with different precipitation values, which may include the following steps: detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb, and the precipitation value of the wheat with the genotype qSV1Ba is greater than the precipitation value of the wheat with the genotype qSV1Bb;

[0009] The wheat of the genotype qSV1Ba is a wheat of CC homozygous genotype based on the C101G SNP site;

[0010] The wheat of the genotype qSV1Bb is a wheat of homozygous GG genotype based on the C101G SNP site;

[0011] The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0012] In the above method, the step of detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb may be as follows:

[0013] (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;

[0014] 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;

[0015] (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.

[0016] 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.

[0017] In the above method, the reaction system for PCR amplification using primer set 1 can be specifically 3.0 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).

[0018] In the above method, the step of detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb may be as follows:

[0019] (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;

[0020] The primer set 2 is composed of the upstream primer F1 shown at the 22nd to 47th positions from the 5' end of SEQ ID NO: 1, the upstream primer F2 shown at the 22nd to 47th positions from the 5' end of SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3;

[0021] (b2) taking the PCR amplification product obtained in step (b1) and sequencing it;

[0022] (b3) Obtaining the genotype of the wheat to be tested according to the sequencing results obtained in step (b2).

[0023] The present invention also protects a kit for identifying or assisting in identifying the precipitation value of wheat. The kit may include a substance for detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb;

[0024] The genotype qSV1Ba is a CC homozygous genotype based on the C101G SNP site;

[0025] The genotype qSV1Bb is based on the C101G SNP site, which is a homozygous GG type;

[0026] The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0027] The kit may specifically be composed of a substance for detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb.

[0028] Any of the above-mentioned substances for detecting whether the genotype of the wheat to be tested is genotype qSV1Ba or genotype qSV1Bb 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.

[0029] Any of the above-mentioned substances for detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb can specifically be primer set 2; the primer set 2 consists of the upstream primer F1 shown at positions 22 to 47 from the 5' end of SEQ ID NO: 1, the upstream primer F2 shown at positions 22 to 47 from the 5' end of SEQ ID NO: 2, and the downstream primer R shown in SEQ ID NO: 3.

[0030] 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.

[0031] The present invention also protects the molecular marker represented by SEQ ID NO: 4 or the 76th to 158th position from the 5' end of SEQ ID NO: 4.

[0032] 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 precipitation value of wheat.

[0033] 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 precipitation values.

[0034] 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.

[0035] In the above, when the genotype of the C101G SNP site in the molecular marker is CC homozygous, the wheat is determined to be of genotype qSV1Ba. When the genotype of the C101G SNP site in the molecular marker is GG homozygous, the wheat is determined to be of genotype qSV1Bb. The C101G SNP site is the 101st nucleotide from the 5' end of SEQ ID NO: 4 in the wheat genome.

[0036] In the above, the precipitation value of wheat of genotype qSV1Ba>the precipitation value of wheat of genotype qSV1Bb.

[0037] In the above, the > may specifically be a statistical >.

[0038] 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.

[0039] 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 C101G SNP site, and the wheat sedimentation value trait can be screened or assisted in screening. 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. A primer combination for screening the wheat sedimentation value is developed based on the difference in the C101G SNP site. The present invention provides an effective detection method for the breeding of new high-quality wheat varieties, which is of great significance to improving the breeding level of high-quality wheat in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The C101G SNP site and the positions of the three primers on the DNA molecule shown in SEQ ID NO:4.

[0041] Figure 2The results of genotyping of 319 wheat germplasms in natural populations (CK is the negative control; GG is the GG homozygous type, i.e., genotype qSV1Bb; CC is the CC homozygous type, i.e., genotype qSV1Ba). DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Example 1. Discovery of the C101G SNP site in the wheat genome and establishment of a wheat genotyping method based on the C101G SNP site

[0046] 1. Discovery of the C101G SNP site in the wheat genome

[0047] After a large number of experiments, the inventors of the present invention found a SNP site in the wheat genome, named C101GSNP site. The C101G SNP site is located at the 101st position from the 5' end of SEQ ID NO: 4, and the genotype is CC homozygous and GG homozygous. In the Chinese spring wheat genome IWGSC RefSeq v1.0, the physical position of the C101G SNP site is the 346408189th position of chromosome 1B; the physical position of the DNA molecule shown in SEQ ID NO: 4 is the 346408089-346408289th position of chromosome 1B.

[0048] 2. Synthesis of primer sets for amplification of C101G SNP locus

[0049] Based on the KASP technical principle, the inventors of the present invention designed and synthesized a primer set for amplifying the C101GSNP 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.

[0050] Table 1

[0051]

[0052] Note: Single underline is FAM fluorescent tag sequence, double underline is HEX fluorescent tag sequence.

[0053] The positions of the C101G 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 C101G SNP site, and the boxes indicate primers).

[0054] 3. Establishment of the genotyping method of wheat based on C101G SNP locus

[0055] After a large number of experiments, the inventors of the present invention established a genotyping method for wheat to be tested based on the C101G SNP site. The specific steps are as follows:

[0056] 1. Obtaining genomic DNA of wheat to be tested

[0057] The genomic DNA of the wheat leaves to be tested was extracted by the CTAB method to obtain the genomic DNA of the wheat to be tested.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The reaction system was 3.0 μL, consisting of 0.75 μL 2×KASP Master Mix (LGC, Lot No. 13426773), 0.0417 μL primer working solution, 1.5 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).

[0062] 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.

[0063] 3. After completing step 2, use a fluorescent signal reader (Omega) and a fluorescent detection system (Araya) to read the fluorescence data of the PCR amplification product and convert the fluorescent signal into an analyzable value. The fluorescence scanning results are graphically displayed using GraphPad Prism, that is, the genotype of the wheat to be tested is determined based on the color of the fluorescent signal. The specific principles are as follows: If the wheat to be tested shows a green fluorescent signal, the genotype of the wheat to be tested based on the C101G SNP site is CC homozygous, and the genotype of the wheat to be tested is named genotype qSV1Ba; if the wheat to be tested shows a pink fluorescent signal, the genotype of the wheat to be tested based on the C101G SNP site is GG homozygous, and the genotype of the wheat to be tested is named genotype qSV1Bb. The negative control has no detection signal.

[0064] The graphical display results are as follows: the genotype qSV1Bb showing a pink fluorescent signal is distributed near the y-axis; the genotype qSV1Ba showing a green fluorescent signal is distributed near the x-axis; and the negative control with no detected signal is distributed near the origin.

[0065] Example 2: Association analysis between two wheat genotypes (genotype qSV1Bb and genotype qSV1Ba) and sedimentation values

[0066] 1. Genotyping of wheat in natural populations

[0067] 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 319 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.

[0068] Test results see Figure 2 The genotypes of the 319 wheat accessions are detailed in column 3 of Table 2. The genotypes of 169 wheat accessions were genotype qSV1Ba, and the genotypes of 150 wheat accessions were genotype qSV1Bb.

[0069] Table 2

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Note: NA means the average sedimentation value data is missing.

[0080] 2. Detection of wheat sedimentation value traits

[0081] 319 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 (2019-2020 and 2020-2021). They were sown every year under normal water and fertilizer conditions (watering 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) under three environments, 3 m row length, randomized block design, three replications. After harvest, 30 grains of each wheat germplasm were randomly selected, and the wheat grains were ground into flour using an experimental mill (Brabender). The sedimentation value of the flour was determined according to the national standard "SDS method for determination of wheat sedimentation index for grain and oil inspection" (GB / T 15685-2011) and the average value was taken, which was recorded as the average sedimentation value.

[0082] The statistical results of the average precipitation values ​​of 319 wheat germplasms under six environments (i.e., normal water and fertilizer in 2019-2020, normal water and fertilizer in 2020-2021, drought treatment in 2019-2020, drought treatment in 2020-2021, low nitrogen treatment in 2019-2020, and low nitrogen treatment in 2020-2021) are shown in columns 4-9 in Table 2. Only some wheat germplasms did not obtain the average precipitation value data due to planting reasons, which are indicated by NA.

[0083] 3. Correlation Analysis

[0084] 1. Take the average sedimentation value of wheat germplasm of the same genotype under the same environment and record it as the mean sedimentation value.

[0085] 2. The two-tailed t-test was used to conduct an association analysis between the wheat germplasm of genotype qSV1Bb and genotype qSV1Ba and the mean sedimentation value in the natural population under the same environment.

[0086] The results are shown in Table 3 (the statistical number of wheat germplasms was obtained based on Tables 2 and 3, i.e., the number of wheat germplasms for which average sedimentation value data was not obtained was removed).

[0087] Table 3

[0088]

[0089] Note: P<0.01 means the difference is extremely significant.

[0090] The results showed that in the natural population composed of 319 wheat germplasms, the precipitation value of wheat with genotype qSV1Ba was greater than that of wheat with genotype qSV1Bb, and the ">" was statistically greater. The study on the natural population showed that genotype qSV1Ba was an excellent genotype for improving the precipitation value of wheat.

[0091] The above results show that the wheat sedimentation value trait can be screened or assisted in screening by detecting the genotype of the wheat to be tested based on the C101G SNP site, which has important application value in the process of wheat molecular marker-assisted breeding.

[0092] 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 precipitation values, comprising the following steps: detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb, and the precipitation value of the wheat with the genotype qSV1Ba is greater than the precipitation value of the wheat with the genotype qSV1Bb; The wheat of the genotype qSV1Ba is a wheat of CC homozygous genotype based on the C101G SNP site; The wheat of the genotype qSV1Bb is a wheat of homozygous GG genotype based on the C101G SNP site; The C101G SNP site is the 101st 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 qSV1Ba or genotype qSV1Bb 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 qSV1Ba or genotype qSV1Bb 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 is composed of the upstream primer F1 shown at the 22nd to 47th positions from the 5' end of SEQ ID NO: 1, the upstream primer F2 shown at the 22nd to 47th positions 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 precipitation value of wheat, comprising a substance for detecting whether the genotype of the wheat to be tested is the genotype qSV1Ba or the genotype qSV1Bb; The genotype qSV1Ba is a CC homozygous genotype based on the C101G SNP site; The genotype qSV1Bb is based on the C101G SNP site, which is a homozygous GG type; The C101G SNP site is the 101st 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 qSV1Ba or genotype qSV1Bb 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 qSV1Ba or genotype qSV1Bb is primer set 2; The primer set 2 consists of an upstream primer F1 shown at positions 22 to 47 from the 5' end of SEQ ID NO: 1, an upstream primer F2 shown at positions 22 to 47 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 76 to 158 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 precipitation value of wheat.

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 auxiliary screening of wheat with different precipitation values.

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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