Molecular marker for regulating and controlling storage protein content of wheat TaB3-2A2 and application of molecular marker

Abstract

The invention discloses a molecular marker of wheat TaB3-2A2 for regulating and controlling storage protein content and application, and belongs to the technical field of molecular markers. In order to solve the technical problem of how to identify wheat with high wheat grain storage protein content or low wheat grain storage protein content, the invention provides application of a primer composition for detecting polymorphism or genotype of an SNP (Single Nucleotide Polymorphism) site in identification or auxiliary identification of the wheat grain storage protein content, the SNP site is an SNP13 site, and the SNP site is a single nucleotide polymorphism (SNP) site. The SNP 13 site is the 71st nucleotide of SEQ ID No.4, and the nucleotide type of the SNP 13 site is T or C. The SNP 13 site is the 71st nucleotide of SEQ ID No.4. The KASP molecular marker has the beneficial effects that the storage protein content of wheat can be predicted by applying the developed KASP molecular marker, so that the cost is saved, the selection efficiency is greatly improved, the breeding process can be accelerated, and a new molecular tool is provided for efficiently screening high-yield wheat varieties and cultivating.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and particularly relates to a molecular marker for regulating the storage protein content in wheat TaB3-2A2 and its application. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the world's three major staple food crops, providing approximately 20% of the energy and protein in the human diet. With the improvement of living standards, people have higher and higher requirements for wheat quality, and quality improvement has become one of the main breeding goals of wheat. The composition and content of wheat grain storage proteins determine the processing characteristics of flour. Improving the composition and content of grain storage proteins is of great significance for wheat processing quality. High-molecular-weight glutenin subunits (HMW-GS) are an important component of wheat storage proteins. As the main component constituting the wheat dough skeleton, they determine the strength and elasticity of the dough. The endosperm-specific high expression of the HMW-GS encoding gene Glu-1 is mainly regulated transcriptionally, which is achieved by the co-action of cis-acting elements in the gene promoter region and transcription factors. Previously, a transcription factor TaB3-2A2 that binds to the Glu-1 promoter was identified using DNA Pulldown combined with liquid chromatography-mass spectrometry technology. Through haplotype and genetic effect analysis of TaB3-2A2, its excellent haplotypes were identified and molecular markers available for breeding were developed, providing a molecular tool for cultivating high-quality and specialized wheat varieties. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: how to identify wheat with high or low wheat grain storage protein content. To solve this technical problem, the present invention provides the following technical solutions:

[0004] The present invention provides the application of a substance for detecting the polymorphism or genotype of SNP sites, and the application is at least one of the following:

[0005] A1) Application in identifying or assisting in identifying the wheat grain storage protein content;

[0006] A2) Application in preparing a product for identifying or assisting in identifying the wheat grain storage protein content;

[0007] A3) Application in screening or assisting in screening wheat varieties with high grain protein content;

[0008] A4) Application in preparing a product for screening or assisting in screening wheat varieties with high grain protein content;

[0009] A5) Application in screening or assisting in screening wheat varieties with low grain protein content;

[0010] A6) Use in the preparation of products for screening or assisting in screening wheat varieties with low grain protein content;

[0011] A7) Use in wheat breeding and / or assisted breeding;

[0012] A8) Use in the preparation of products for wheat breeding and / or assisted breeding;

[0013] The SNP locus is the SNP13 locus, which is a SNP locus in the wheat genome, such as the 71st nucleotide of SEQ ID No. 4, and its nucleotide type is T or C.

[0014] The genotype of the SNP13 locus can be TT, CC or TC. The TT is the homozygous type with the nucleotide type of T at the SNP13 locus in the wheat genome; the CC genotype represents the homozygous type with the nucleotide type of C at the SNP13 locus in the wheat genome; the TC genotype represents the heterozygous type with the nucleotide types of T and C at the SNP13 locus in the wheat genome.

[0015] The grain protein content of wheat with the CC genotype at the SNP13 locus in the wheat genome is higher than or candidate higher than that of wheat with the TT genotype and / or TC genotype at the SNP13 locus in the wheat genome.

[0016] The grain protein content of wheat with the TT genotype at the SNP13 locus in the wheat genome is lower than or candidate lower than that of wheat with the CC genotype and / or TC genotype at the SNP13 locus in the wheat genome.

[0017] In this application, the wheat to be tested can be a pure line or an inbred line. The inbred line can be a recombinant inbred line.

[0018] Furthermore, the substance for detecting the polymorphism or genotype of the SNP locus is a primer composition for amplifying a wheat genomic DNA fragment including the SNP13 locus.

[0019] Furthermore, the primer composition is composed of a single-stranded DNA with the nucleotide sequence of positions 22 - 39 of SEQ ID No. 1, a single-stranded DNA with the nucleotide sequence of positions 22 - 39 of SEQ ID No. 2, and a single-stranded DNA with the nucleotide sequence of SEQ ID No. 3.

[0020] In the present application, the PCR primers in the primer composition may or may not be labeled with a labeling agent. The labeling agent refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Labeling agents include, but are not limited to, dyes; radioactive labels such as 32P; binding moieties such as biotin; haptens such as digoxin (DIG); luminescent, phosphorescent, or fluorescent moieties; and individual fluorescent dyes or fluorescent dyes combined with moieties that can inhibit or shift the emission spectrum through fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or, optionally, can be charge-neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

[0021] Further, the primer composition consists of the single-stranded DNA shown in SEQ ID No.1, the single-stranded DNA shown in SEQ ID No.2, and the single-stranded DNA shown in SEQ ID No.3.

[0022] In the single-stranded DNA shown in SEQ ID No.1 in the present application, positions 1-21 of SEQ ID No.1 are the specific recognition sequences for the FAM fluorescent probe. In the single-stranded DNA shown in SEQ ID No.2, positions 1-21 of SEQ ID No.1 are the specific recognition sequences for the HEX fluorescent probe.

[0023] The present invention also provides the above-mentioned primer composition.

[0024] The present invention also provides a reagent and / or kit containing the above-mentioned primer composition.

[0025] The present invention also provides the application of the reagent and / or kit in at least one of the following:

[0026] B1) Application in identifying or assisting in identifying the content of wheat grain storage proteins;

[0027] B2) Application in screening or assisting in screening wheat varieties with high grain protein content;

[0028] B3) Application in screening or assisting in screening wheat varieties with low grain protein content;

[0029] B4) Application in wheat breeding and / or assisting in breeding.

[0030] The present invention also provides a DNA molecule, and the nucleotide sequence of the DNA molecule is the DNA molecule shown in SEQ ID No.4.

[0031] The present invention also provides a method for identifying or assisting in identifying the storage protein content of wheat grains. The method includes using a substance for detecting the polymorphism or genotype of the SNP13 locus to detect the genotype of the SNP13 locus of the wheat to be tested, and identifying or assisting in identifying the storage protein content of the wheat grains according to the polymorphism or genotype of the SNP13 locus of the wheat to be tested;

[0032] The SNP13 locus is a SNP locus in the wheat genome, which is the 71st nucleotide of SEQ ID No. 4, and the nucleotide type thereof is T or C.

[0033] Furthermore, the method for detecting the genotype of the SNP13 locus of the wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, performing PCR amplification with the primer composition, and obtaining a PCR product; determining the polymorphism or genotype of the SNP13 locus according to the sequencing result or fluorescence signal of the PCR product.

[0034] Furthermore, the grain protein content of wheat with the CC genotype at the SNP13 locus in the wheat genome is higher than or candidate higher than that of wheat with the TT genotype and / or TC genotype at the SNP13 locus in the wheat genome.

[0035] Furthermore, the grain protein content of wheat with the TT genotype at the SNP13 locus in the wheat genome is lower than or candidate lower than that of wheat with the CC genotype and / or TC genotype at the SNP13 locus in the wheat genome.

[0036] The present invention also provides a method for wheat breeding, which includes any one of the following:

[0037] C1) Selecting wheat with the TT genotype at the SNP13 locus as a parent for breeding. The TT genotype represents a homozygous type in which the nucleotide type of the SNP13 locus in the wheat genome is T. The purpose of the breeding includes cultivating wheat with a grain protein content higher than that of the parent;

[0038] C2) Selecting wheat with the CC genotype at the SNP13 locus as a parent for breeding. The CC genotype represents a homozygous type in which the nucleotide type of the SNP13 locus in the wheat genome is C. The purpose of the breeding includes cultivating wheat with a grain protein content higher than that of the parent.

[0039] The competitive allele-specific PCR (KASP) provided by the present invention comprises using the genomic DNA of the wheat to be tested as a template, and using the above primer composition as the PCR reaction primer to perform a PCR amplification reaction to harvest the amplification product. When the temperature of the PCR amplification product drops below 40°C, the fluorescence values are read by scanning with the FAM and HEX beams of a microplate reader (the FAM fluorescent label is observed and read at the excitation wavelength of 485 nm and the emission wavelength of 520 nm, and the HEX fluorescent label is observed and read at the excitation wavelength of 528 nm and the emission wavelength of 560 nm). The genotype of the wheat to be tested based on the SNP13 locus is judged according to the color of the fluorescence signal as follows: If the wheat to be tested shows a blue fluorescence signal based on the SNP13 marker, the genotype of the SNP13 locus of the wheat to be tested is the TT homozygous type; if the wheat to be tested shows a red fluorescence signal based on the SNP13 marker, the genotype of the SNP13 locus of the wheat to be tested is the CC homozygous type.

[0040] The allelic variant bases of TaB3-2A2-Hap1 haplotype at positions 88982491, 88982578, 88982609, 88982782, 88982827, 88982942, 88982997, 88983009, 88983055, 88983100, 88983159, 88983293, 88983810 and 88988219, 88988484, 88988516 and 88988526 in the Chinese Spring wheat reference genome sequence RefSeq v1.0 are A, A, T, C, A, G, A, C, C, A, G, A, T, TA, G, T and A respectively.

[0041] The allelic variant bases of TaB3-2A2-Hap2 haplotype at positions 88982491, 88982578, 88982609, 88982782, 88982827, 88982942, 88982997, 88983009, 88983055, 88983100, 88983159, 88983293, 88983810 and 88988219, 88988484, 88988516 and 88988526 in the Chinese Spring wheat reference genome sequence RefSeq v1.0 are G, G, C, CG, AAGG, A, G, G, T, AC, A, C, C, T, C, C and G respectively.

[0042] In the TaB3-2A2-Hap1 haplotype and TaB3-2A2-Hap2 haplotype, 17 variant sites are in linkage relationship. According to the SNP13 site genotype divided by the above KASP marker, the TaB3-2A2 gene can be divided into two haplotypes: the wheat to be tested with the genotype TT at the SNP13 site is the TaB3-2A2-Hap1 haplotype; the wheat to be tested with the genotype CC at the SNP13 site is the TaB3-2A2-Hap2 haplotype.

[0043] The beneficial technical effects achieved by the present invention are as follows:

[0044] By applying the KASP molecular marker developed by the present invention, the storage protein content of wheat can be predicted, which not only saves costs, but also greatly improves the selection efficiency, can accelerate the breeding process, and provides new possibilities for the efficient screening and cultivation of high-yield wheat varieties.

[0045] The primer composition for detecting the SNP13 site genotype or polymorphism provided by the present invention was used to genotype 138 lines from the Huanghuai wheat region of China. Genetic effect analysis found that the lines with the genotype CC at the SNP13 site had a significantly increased protein content compared with the TaB3-2A2-Hap1 lines with the genotype TT at the SNP13 site, and the yield did not decrease significantly. Considering that there is generally a negative correlation between the storage protein content of wheat grains and the yield, the TaB3-2A2-Hap2 with the genotype CC at the SNP13 site significantly improves the protein content without significantly reducing the yield, which coordinates the imbalance between the grain protein content and the yield to a certain extent. Therefore, this haplotype is an excellent allelic variation for breeding high-quality strong gluten wheat. Description of the Drawings

[0046] Figure 1 are the variant sites and main haplotypes of wheat TaB3-2A2;

[0047] Figure 2 is the genotyping of TaB3-2A2 of wheat lines in the Huanghuai wheat region using KASP markers. Detailed Embodiments

[0048] The terms in this application:

[0049] Examples of resources that describe many of the terms related to molecular biology used herein can be found in the following references: Alberts et al., Molecular Biology of The Cell, 5th Edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Edition, Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th Edition, Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0050] Any reference cited herein, including for example all patents, published patent applications, and non-patent publications, is hereby incorporated by reference in its entirety.

[0051] For the purposes of facilitating understanding of the present disclosure, several terms and abbreviations used herein are defined as follows:

[0052] When used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either A or B or both, i.e., A alone, B alone, or a combination of A and B. The expression "A, B, and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0053] The term "gene" refers to a DNA segment involved in the production of a polypeptide chain; it includes regions (leader region and trailer region) before and after the coding region involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as the intervening sequences (introns) between individual coding regions (exons).

[0054] The term "allele" refers to one of the multiple alternative forms of a gene or non-coding region of DNA that occupy the same position on a chromosome. The term allele can be used to describe DNA from any organism, including but not limited to bacteria, viruses, fungi, protozoa, molds, yeasts, plants, humans, non-human animals, and archaea.

[0055] The term "genotype" refers to the total genetic combination of a biological individual. Biological individuals include, but are not limited to, diploids, tetraploids, or other possible polyploids. For example, in diploids, the genotype can be homozygous or heterozygous. A homozygous genotype means that the two alleles are exactly the same, such as AA or aa. A heterozygous type means that the two alleles are different, such as Aa. Other polyploid genotypes can occur and be described according to the actual situation of the alleles.

[0056] The term "template" refers to any nucleic acid molecule that can be used for the amplification described in the present invention. RNA or DNA that is not a natural double strand can be made into double-stranded DNA and thus used as double-stranded DNA. Any double-stranded DNA or product containing a variety of different double-stranded DNA molecules can be used as template DNA to amplify one or more loci contained in the template DNA.

[0057] The term "primer" refers to an oligonucleotide that can be used in amplification methods such as polymerase chain reaction (PCR), which is used to amplify a nucleotide sequence according to a polynucleotide sequence corresponding to a specific genomic sequence. At least one pair of PCR primers for amplifying a polynucleotide sequence is sequence-specific for that sequence.

[0058] The term "amplification reaction" refers to a process for copying nucleic acids one or more times. In embodiments, the amplification methods include, but are not limited to: polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-β phage amplification, strand displacement amplification, or overlap extension splicing polymerase chain reaction. In some embodiments, single-molecule nucleic acids are amplified, for example, by digital PCR.

[0059] Terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0060] The terms "storage protein content", "grain storage protein content", and "seed protein content" have the same meaning, specifically referring to the total protein content of wheat grains.

[0061] The technical solutions provided by this application:

[0062] The following further describes this application in detail in combination with specific embodiments. The examples given are only for clarifying this application, rather than limiting the scope of this application. The following examples provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit this application in any way.

[0063] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0064] 138 natural populations in the Huanghuai wheat region in the following examples have been recorded in: Li J, Xie L, Tian X, Liu S, Xu D, Jin H, Song J, Dong Y, Zhao D, Li G, Li Y, Zhang Y, Zhang Y, Xia X, He Z, Cao S. (2021) TaNAC100 acts as an integrator of seed protein and starch synthesis exerting pleiotropic effects on agronomic traits in wheat. Plant Journal 108(3):829-840. The biological material can be obtained from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0065] In the quantitative tests in the following examples, unless otherwise specified, three replicates are set, and the results are averaged. The following examples use Excel statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and t-test is used. P < 0.05 (*) indicates significant difference.

[0066] Example 1: Identification of polymorphic sites and haplotypes of wheat TaB3-2A2 gene

[0067] The wheat genome ID (TraesCS2A02G144100) of the transcription factor encoding gene TaB3-2A2 was input into the wheat genome variation database (Wheat-SnpHub-Portal, http: / / wheat.cau.edu.cn / Wheat_SnpHub_Portal / ). 188 varieties (MP group and NC-CC group) in the database were selected, and the variation information of 1Kb upstream and downstream of the gene and the gene open reading frame was retrieved. A total of 17 variation sites were found, denoted as SNP1, SNP2, SNP3, Indel4, Indel5, SNP6, SNP7, SNP8, SNP9, Indel10, SNP11, SNP12, SNP 13, Indel14, SNP15, SNP16 and SNP17 ( Figure 1), the physical positions of these 17 mutation sites in the Chinese Spring wheat reference genome sequence RefSeq v1.0 are 88982491, 88982578, 88982609, 88982782, 88982827, 88982942, 88982997, 88983009, 88983055, 88983100, 88983159, 88983293, 88983810, 88988219, 88988484, 88988516, and 88988526, respectively. These mutation information forms two haplotypes ( Figure 1 ). Among them, the allelic variant bases of TaB3-2A2-Hap1 at the 88982491st, 88982578th, 88982609th, 88982782nd, 88982827th, 88982942nd, 88982997th, 88983009th, 88983055th, 88983100th, 88983159th, 88983293rd, 88983810th, 88988219th, 88988484th, 88988516th, and 88988526th positions in the Chinese Spring wheat reference genome sequence RefSeq v1.0 are A, A, T, C, A, G, A, C, C, A, G, A, T, TA, G, T, and A, respectively; the allelic variant bases of TaB3-2A2-Hap2 at the 88982491st, 88982578th, 88982609th, 88982782nd, 88982827th, 88982942nd, 88982997th, 88983009th, 88983055th, 88983100th, 88983159th, 88983293rd, 88983810th, 88988219th, 88988484th, 88988516th, and 88988526th positions in the Chinese Spring wheat reference genome sequence RefSeq v1.0 are G, G, C, CG, AAGG, A, G, G, T, AC, A, C, C, T, C, C, and G, respectively. The 17 mutation sites in TaB3-2A2-Hap1 haplotype and TaB3-2A2-Hap2 haplotype are in a linkage relationship.

[0068] Example 2. Development of molecular markers for differentiating and identifying TaB3-2A2 haplotypes and their specific primer compositions

[0069] Competitive allele-specific PCR (KASP) molecular markers can be used for selection targeting target traits at the DNA level. They not only have accurate genotyping but also high throughput and low cost, making them one of the most suitable molecular marker technologies for assisting breeding. Through a large number of experiments, the inventors of the present invention designed a set of primer sets K-2A-SNP13 suitable for KASP to identify the TaB3-2A2 haplotype in wheat at SNP13 on chromosome 2A (position 88983810 in the Chinese Spring wheat reference genome sequence RefSeq v1.0) with the help of Polymarker (https: / / www.polymarker.info / ), and evaluated the primer sequence specificity using WheatOmics (http: / / 202.194.139.32 / ).

[0070] The KASP primer set consists of forward primers F1 and F2 and a reverse primer R.

[0071] For SNP13 (position 88983810 in the Chinese Spring wheat reference genome sequence RefSeq v1.0), the following primer set was designed:

[0072] Forward primer F1: 5′-gaaggtgaccaagttcatgctTGTCGCCCAAAACATCGT-3′ (the lowercase letter part is the specific recognition sequence SEQ ID No.1 of the FAM fluorescent probe);

[0073] Forward primer F2: 5′-gaaggtcggagtcaacggattTGTCGCCCAAAACATCGC-3′ (the lowercase letter part is the specific recognition sequence SEQ ID No.2 of the HEX fluorescent probe);

[0074] Reverse primer R: 5′-CCATGGTAGTGGTATGCGCA-3′ (SEQ ID No.3).

[0075] The K-2A-SNP13 marker can identify whether the nucleotide at the 71st position from the 5′ end of SEQ ID No.4 in the wheat genome (corresponding to the last base at the 3′ end of the two forward primers) is T or C, which is represented by y in SEQ ID No.4.

[0076] SEQ ID No.4: 5′-GCAAAGCTAGACCCCATCCAGAGCATCATCTGCCTCTGCA TACTGAACATCCATGTCGCCCAAAACATCGyCCGTCTGCGGTCGAGACCGAAGC TGTGCGCATACCACTACCATGGTGCGGTTGCTAACCGCCGCAGCATC-3′。

[0077] Based on the genotypes of SNP13 locus divided by the above KASP markers, the TaB3-2A2 gene can be divided into two haplotypes: the genotype of TaB3-2A2-Hap1 at SNP13 is TT; the genotype of TaB3-2A2-Hap2 at SNP13 is CC.

[0078] Example 3: Application of Molecular Marker K-2A-SNP13 and Its Specific Primer Set in Identifying the Haplotypes of TaB3-2A2 in Natural Population of Wheat

[0079] 1. Field Phenotypic Identification and Data Analysis of 138 Natural Populations in the Yellow and Huai River Wheat Regions

[0080] 138 natural wheat varieties in the Yellow and Huai River wheat regions were planted in Anyang, Henan and Suixi, Anhui in the 2012 - 2013 and 2013 - 2014 growing seasons, and in Anyang, Henan and Gaoyi, Hebei in the 2014 - 2015 growing season. A completely randomized block design was used with three replicates, single-row plots, plot length of 1.5 m, row width of 0.2 m, and 50 seeds per row. Field management measures were carried out according to the local wheat field management specifications.

[0081] Phenotypic data of yield-related traits were provided by Li Jihu of this laboratory as described above (Li et al., 2021). The content of grain storage protein was analyzed using a near-infrared reflectance spectrometer (Perten DA 7200, Spring-field, IL, USA). The specific detection steps included: 1. Sample treatment: The wheat grain samples to be tested were cleaned to remove bran, damaged grains and other impurities to avoid affecting the analysis results; 2. Sample loading: The wheat samples to be tested were evenly loaded into the sample box to ensure a flat sample surface and avoid light scattering and reflection interference; 3. Spectral scanning: The samples were scanned using a near-infrared analyzer to collect spectral data in the near-infrared region. Each sample was usually scanned multiple times and the average value was taken to improve the reliability of the data. The total protein content in the wheat grains to be tested obtained was the content of grain storage protein. The BLUP values of the phenotypic data of grain storage protein content and yield traits are shown in Table 1.

[0082] 2. Detection of Genotypes of Lines in the Yellow and Huai River Wheat Regions by Molecular Marker K-2A-SNP13

[0083] (1) Genomic DNA was extracted from the young leaves of 138 wheat lines using the CTAB method.

[0084] Both the quality and concentration of the genomic DNA must meet the requirements of PCR. The standards are as follows: Agarose gel electrophoresis shows a single DNA band without obvious smearing; The ratio of A260 / A280 detected by the ultraviolet spectrophotometer Nanodrop2100 (Thermo) is between 1.8 - 2.0 (no protein contamination in the DNA sample), the ratio of A260 / A230 is between 1.8 - 2.0 (low salt ion concentration in the DNA sample), and there is no obvious light absorption at 270 nm (no phenol contamination in the DNA sample); The concentration of the wheat genomic DNA to be tested is between 50 - 200 ng / μL.

[0085] (2) Kompetitive allele - specific PCR (KASP)

[0086] Using the genomic DNA of the wheat to be tested as a template, PCR amplification was carried out with the KASP primer set synthesized in Example 2 to obtain amplification products. Reaction system: 2.0 μL KASP 2×Master Mix (LGC, product number: 13448166), 0.048 μL KASP primer (a mixture of 3 primers with a total concentration of 50 μM, and the molar ratio of two forward primers to one reverse primer is 2:2:5), 2.0 μL template DNA (50 ng / μL). The reaction program was: pre - denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61°C - 55°C (touch - down program was selected, decreasing by 0.6°C per cycle), 1 min, for 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 1 min, and continue to amplify for 31 cycles.

[0087] (3) Fluorescence signal acquisition and genotyping

[0088] Fluorescence signals were detected on a PHERAstar Plus automated fluorescence multi-functional microplate reader (BMG Labtech GmbH, Ortenberg, Germany), and genotyping was performed using KlusterCaller software (LGC, Hoddesdon, UK). When the temperature of the PCR amplification product dropped below 40 °C, fluorescence values were read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescence label was observed and read at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, and the HEX fluorescence label was observed and read at an excitation wavelength of 528 nm and an emission wavelength of 560 nm). The genotype of the wheat to be tested at the SNP13 locus was determined based on the color of the fluorescence signal. The specific judgment principles are as follows: If the wheat to be tested shows a blue fluorescence signal based on the K-2A-SNP13 marker, the genotype of the SNP13 locus of the wheat to be tested is the TT homozygous type; if the wheat to be tested shows a red fluorescence signal based on the K-2A-SNP13 marker, the genotype of the SNP13 locus of the wheat to be tested is the CC homozygous type.

[0089] According to the linkage relationship of the 17 variant sites described in Example 1, the wheat to be tested with the genotype TT at SNP13 is the TaB3-2A2-Hap1 haplotype. The wheat to be tested with the genotype CC at SNP13 is the TaB3-2A2-Hap2 haplotype.

[0090] The haplotype classification of 138 wheat lines is shown in Table 1, and the specific genotyping results are shown in Figure 2 。

[0091] 3. Association analysis of TaB3-2A2 gene haplotypes with storage protein content and yield in wheat

[0092] According to the genotyping results and phenotypic data, a t-test was performed using the TTEST model of Excel statistical software to determine the genetic effects of different TaB3-2A2 haplotypes on storage protein content and yield. The genetic analysis results of storage protein content and yield are shown in Table 2, and the experimental results are expressed as mean ± standard deviation.

[0093] Table 1. Haplotype classification and phenotypic data of 138 representative wheat lines in the Huanghuai wheat region using the molecular marker K-2A-SNP13

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] Note: For the wheat to be tested with a homozygous genotype of T at the SNP13 locus, the genotype of SNP13 is represented by TT, and this wheat to be tested is of the TaB3-2A2-Hap1 haplotype, denoted as Hap1 in the table; for the wheat to be tested with a homozygous genotype of C at the SNP13 locus, the genotype of SNP13 is represented by CC, and this wheat to be tested is of the TaB3-2A2-Hap2 haplotype, denoted as Hap2 in the table; SNP13 is a SNP locus in the wheat genome, which is the 71st nucleotide of SEQ ID N0.4, and its nucleotide type is T or C. The wheat to be tested are all homozygous wheat lines.

[0101] Table 2. Analysis of the phenotypic relationship between TaB3-2A2 gene haplotypes and wheat protein content and yield-related traits

[0102]

[0103] Note: Table 2 shows the statistical comparison of storage protein content and yield traits between two genotypes, TaB3-2A2-Hap1 and TaB3-2A2-Hap2. *, indicates significant difference (P<0.05).

[0104] In summary, the haplotype analysis results show that:

[0105] (1) Compared with the TaB3-2A2-Hap1 type, the protein content of TaB3-2A2-Hap2 increased significantly by 3.04%;

[0106] (2) Compared with the TaB3-2A2-Hap1 type, the yield of TaB3-2A2-Hap2 decreased by 2.48%, but the decrease was not significant.

[0107] The following conclusions are drawn:

[0108] The grain protein content of the wheat to be tested with the CC genotype (i.e., TaB3-2A2-Hap2 haplotype) at the SNP13 locus in the wheat genome is higher than or candidate higher than that of the wheat to be tested with the TT genotype (i.e., TaB3-2A2-Hap1 haplotype) at the SNP13 locus in the wheat genome.

[0109] The grain protein content of the wheat to be tested with the TT genotype (i.e., TaB3-2A2-Hap1 haplotype) at the SNP13 locus in the wheat genome is lower than or candidate lower than that of the wheat to be tested with the CC genotype (i.e., TaB3-2A2-Hap2 haplotype) at the SNP13 locus in the wheat genome.

[0110] The above has described the present application in detail. For those skilled in the art, without departing from the spirit and scope of the present application and without the need for unnecessary experiments, the present application can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present application are given, it should be understood that the present application can be further improved. In short, according to the principle of the present application, the present application is intended to cover any modifications, uses, or improvements to the present application, including those that depart from the scope disclosed in the present application but are made by using conventional techniques known in the art.

Claims

1. Use of a substance for detecting the polymorphism or genotype of an SNP locus, characterized in that, The application is at least one of the following: A1) Application in identifying or assisting in identifying the storage protein content of wheat grains; A2) Application in preparing a product for identifying or assisting in identifying the storage protein content of wheat grains; A3) Application in screening or assisting in screening wheat varieties with high grain protein content; A4) Application in preparing a product for screening or assisting in screening wheat varieties with high grain protein content; A5) Application in screening or assisting in screening wheat varieties with low grain protein content; A6) Application in preparing a product for screening or assisting in screening wheat varieties with low grain protein content; A7) Application in wheat breeding and / or assisting in wheat breeding; A8) Application in preparing a product for wheat breeding and / or assisting in wheat breeding; The SNP locus is the SNP13 locus, and the SNP13 locus is an SNP locus in the wheat genome, such as the 71st nucleotide of SEQ ID No. 4, and the nucleotide type thereof is T or C.

2. The application according to claim 1, wherein The substance for detecting the polymorphism or genotype of the SNP locus is a primer composition for amplifying a wheat genomic DNA fragment including the SNP13 locus.

3. The application according to claim 2, characterized in that, The primer composition consists of a single-stranded DNA with a nucleotide sequence of positions 22-39 of SEQ ID No. 1, a single-stranded DNA with a nucleotide sequence of positions 22-39 of SEQ ID No. 2, and a single-stranded DNA with a nucleotide sequence of SEQ ID No.

3.

4. The application according to claim 2 or 3, characterized in that, The primer composition consists of the single-stranded DNA shown in SEQ ID No. 1, the single-stranded DNA shown in SEQ ID No. 2, and the single-stranded DNA shown in SEQ ID No.

3.

5. The primer composition according to any one of claims 2-4 and / or a reagent and / or kit containing the primer composition.

6. Application of the reagent and / or kit according to claim 5 in at least one of the following: B1) Application in identifying or assisting in identifying the storage protein content of wheat grains; B2) Application in screening or assisting in screening wheat varieties with high grain protein content; B3) Application in screening or assisting in screening wheat varieties with low grain protein content; B4) Application in wheat breeding and / or assisting in wheat breeding.

7. A DNA molecule, wherein the DNA molecule is a DNA molecule with a nucleotide sequence shown in SEQ ID No.

4.

8. A method for identifying or assisting in the identification of the storage protein content in wheat grains, characterized in that, The method includes using a substance for detecting the polymorphism or genotype of the SNP13 locus to detect the genotype of the SNP13 locus of the wheat to be tested, and identifying or assisting in identifying the storage protein content of the wheat grains according to the polymorphism or genotype of the SNP13 locus of the wheat to be tested; The SNP13 locus is an SNP locus in the wheat genome, which is the 71st nucleotide of SEQ ID No. 4, and the nucleotide type thereof is T or C.

9. The method according to claim 8, wherein The method for detecting the genotype of the SNP13 locus of the wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, and performing PCR amplification using the primer composition according to claim 5 to obtain a PCR product; Determine the polymorphism or genotype of the SNP13 locus according to the sequencing result or fluorescence signal of the PCR product.

10. A method for wheat breeding, characterized in that, The method includes any one of the following: C1) Selecting wheat with the genotype of TT at the SNP13 locus described in claim 1 as a parent for breeding, where the TT genotype represents a homozygous type with the nucleotide type of T at the SNP13 locus in the wheat genome; C2) Selecting wheat with the genotype of CC at the SNP13 locus described in claim 1 as a parent for breeding, where the CC genotype represents a homozygous type with the nucleotide type of C at the SNP13 locus in the wheat genome.

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