Molecular marker of wheat ta b3-2a2 regulating storage protein content and application

Abstract

The application discloses a molecular marker for regulating storage protein content of wheat TaB3-2A2 and application thereof, and belongs to the technical field of molecular markers. The technical problem to be solved by the application is how to identify wheat with high grain storage protein content or low grain content. To solve the technical problem, the application provides application of a primer composition for detecting polymorphism or genotypes of an SNP site in identification or auxiliary identification of grain storage protein content of wheat, the SNP site is an SNP13 site, the SNP13 site is a 71th nucleotide in SEQ ID No. 4, and the nucleotide type is T or C. The application has the advantages that the KASP molecular marker developed by the application can be used to predict the storage protein content of wheat, cost is saved, selection efficiency is greatly improved, the breeding process is accelerated, and a new molecular tool is provided for efficient screening of high-yield wheat varieties and cultivation.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to molecular markers and their applications for regulating the content of storage proteins in wheat TaB3-2A2. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of the world's three major staple crops, providing approximately 20% of the energy and protein in the human diet. With rising living standards, people have increasingly higher demands for wheat quality, making quality improvement one of the main breeding goals for wheat. The composition and content of wheat grain storage proteins determine the processing characteristics of flour, and improving the composition and content of grain storage proteins is of great significance to wheat processing quality. High molecular weight glutenin (HMW-GS) is an important component of wheat storage proteins. As a major component constituting the backbone of wheat dough, it determines the strength and elasticity of the dough. The endosperm-specific high expression of the HMW-GS encoding gene Glu-1 is mainly regulated by transcription, achieved through the combined action of cis-acting elements in the gene promoter region and transcription factors. Previously, DNA pulldown combined with liquid chromatography-mass spectrometry (LC-MS) technology was used to identify a transcription factor, TaB3-2A2, that binds to the Glu-1 promoter. Through haplotype and genetic effect analysis of TaB3-2A2, its superior haplotype was identified, and molecular markers usable for breeding were developed, providing molecular tools for breeding high-quality specialty wheat varieties. Summary of the Invention

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

[0004] This invention provides an application of a substance for detecting polymorphisms or genotypes at SNP sites, wherein the application is at least one of the following:

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

[0006] A2) Application in the preparation of products for identifying or assisting in the identification of 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 the preparation of products for screening or assisting in the screening of 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) Application in the preparation of products for screening or assisting in the screening of wheat varieties with low grain protein content;

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

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

[0013] The SNP site is SNP13, which is an SNP site 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. TT is a homozygous type of SNP13 locus in the wheat genome with nucleotide type T. CC genotype represents a homozygous type of SNP13 locus in the wheat genome with nucleotide type C. TC genotype represents a heterozygous type of SNP13 locus in the wheat genome with nucleotide types T and C.

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

[0016] The grain protein content of wheat with the TT genotype at SNP13 in the wheat genome is lower or candidate lower than that of wheat with the CC genotype and / or TC genotype at SNP13 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 used to detect the polymorphism or genotype of the SNP site is a primer composition for amplifying wheat genomic DNA fragments including the SNP13 site.

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

[0020] In this application, the PCR primers in the primer composition may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32p; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum via fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moieties (positive or negative) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).

[0021] Furthermore, 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 of this application, positions 1-21 of SEQ ID No. 1 are the specific recognition sequence of 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 sequence of the HEX fluorescent probe.

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

[0024] The present invention also provides reagents and / or kits comprising the above-described primer compositions.

[0025] This invention also provides the use of the reagents and / or kits in at least one of the following:

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

[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 assisted breeding.

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

[0031] The present invention also provides a method for identifying or assisting in the identification of wheat grain storage protein content, the method comprising using a substance for detecting the polymorphism or genotype of the SNP13 site to detect the genotype of the SNP13 site of the wheat to be tested, and identifying or assisting in the identification of wheat grain storage protein content based on the polymorphism or genotype of the SNP13 site of the wheat to be tested.

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

[0033] Furthermore, the method for detecting the genotype of the SNP13 site in the wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, performing PCR amplification using the primer composition to obtain PCR products; and determining the polymorphism or genotype of the SNP13 site based on the sequencing results or fluorescence signal of the PCR products.

[0034] Furthermore, wheat with the CC genotype at SNP13 in the wheat genome has a higher or candidate higher grain protein content than wheat with the TT and / or TC genotypes at SNP13 in the wheat genome.

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

[0036] This invention also provides a method for wheat breeding, the method comprising any one of the following:

[0037] C1) Select wheat with the genotype TT at the SNP13 locus as the parent for breeding. The TT genotype represents the homozygous type of nucleotide T at the SNP13 locus in the wheat genome. The purpose of the breeding is to cultivate wheat with a higher grain protein content than the parent.

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

[0039] The competitive allele-specific PCR (KASP) provided by this invention includes using wheat genomic DNA as a template, using the above-mentioned primer composition as PCR primers to perform PCR amplification reaction and harvesting the amplification product. When the temperature of the PCR amplification product drops below 40°C, the fluorescence value is read by scanning with FAM and HEX beams of an enzyme-linked immunosorbent assay (ELISA) reader (FAM fluorescent tags are read at excitation wavelengths of 485 nm and emission wavelengths of 520 nm, and HEX fluorescent tags are read at excitation wavelengths of 528 nm and emission wavelengths of 560 nm). The genotype of the wheat at the SNP13 locus is determined based on the fluorescence signal color as follows: if the wheat at the SNP13 locus shows a blue fluorescence signal, the genotype of the wheat at the SNP13 locus is TT homozygous; if the wheat at the SNP13 locus shows a red fluorescence signal, the genotype of the wheat at the SNP13 locus is CC homozygous.

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

[0041] Haplotype TaB3-2A2-Hap2 was found in the Chinese spring wheat reference genome sequence RefSeq. The allelic variations at positions 88982491, 88982578, 88982609, 88982782, 88982827, 88982942, 88982997, 88983009, 88983055, 88983100, 88983159, 88983293, 88983810, 88988219, 88988484, 88988516, and 88988526 in 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 and TaB3-2A2-Hap2 haplotypes, 17 variant sites are linked. Based on the SNP13 locus genotypes identified by the KASP markers, the TaB3-2A2 gene can be divided into two haplotypes: the wheat sample with the SNP13 locus genotype TT is the TaB3-2A2-Hap1 haplotype; the wheat sample with the SNP13 locus genotype CC is the TaB3-2A2-Hap2 haplotype.

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

[0044] The KASP molecular marker developed by this invention can predict the storage protein content of wheat, which not only saves costs but also greatly improves selection efficiency, accelerates the breeding process, and provides new possibilities for efficient screening and breeding of high-yield wheat varieties.

[0045] Using the primer composition provided in this invention for detecting the genotype or polymorphism of SNP13, genotyping was performed on 138 wheat lines from the Huang-Huai wheat region of my country. Genetic effect analysis revealed that the line with the SNP13 genotype CC had a significantly increased protein content compared to the line with the SNP13 genotype TT (TaB3-2A2-Hap1), without a significant decrease in yield. Considering that wheat grain storage protein content is generally negatively correlated with yield, the fact that TaB3-2A2-Hap2 with the SNP13 genotype CC significantly increased protein content without a significant decrease in yield, to some extent reconciles the imbalance between grain protein content and yield. Therefore, this haplotype represents an excellent allelic variant for breeding high-quality, strong-gluten wheat. Attached Figure Description

[0046] Figure 1 The variant sites and major haplotypes of wheat TaB3-2A2;

[0047] Figure 2 To use KASP markers to genotype the TaB3-2A2 wheat variety in the Huang-Huai wheat region. Detailed Implementation

[0048] Terminology in this application:

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

[0050] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0051] For ease of understanding of this 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 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 or both of A and B, 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 segment of DNA involved in the production of a polypeptide chain; it includes regions before and after the coding region (leader and tail regions) involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as insertion sequences (introns) between individual coding regions (exons).

[0054] The term "allele" refers to one of several alternative forms of a gene or non-coding region of DNA that occupies the same location 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-humans, animals, and archaea.

[0055] The term "genotype" refers to the total combination of all genes in an organism. Organisms 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 identical, such as AA or aa. A heterozygous genotype means that the two alleles are different, such as Aa. Other polyploid genotypes can be described based on the specific allele distribution.

[0056] The term "template" refers to any nucleic acid molecule that can be used for the amplification described in this invention. Non-natural double-stranded RNA or DNA can be made into double-stranded DNA for use as double-stranded DNA. Any double-stranded DNA or preparation containing a variety of different double-stranded DNA molecules can be used as template DNA to amplify one or more loci contained within the template DNA.

[0057] The term "primer" refers to an oligonucleotide that can be used in amplification methods such as polymerase chain reaction (PCR) to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a specific genomic sequence. At least one PCR primer used to amplify the polynucleotide sequence is sequence-specific to that sequence.

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

[0059] The terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are 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 solution provided in this application is:

[0062] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] The 138 natural populations of wheat from the Huang-Huai region in the following examples have been described 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, exercising pleiotropic effects on agronomic traits in wheat. Plant Journal 108(3):829-840. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0065] Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Data were processed using Excel statistical software, and experimental results are expressed as mean ± standard deviation. A t-test was used, and P < 0.05 (*) indicated a significant difference.

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

[0067] The wheat genome ID (TraesCS2A02G144100) of the transcription factor encoding gene TaB3-2A2 was entered 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) were selected from the database, and variation information of the gene's upstream and downstream 1Kb and open reading frames was retrieved. A total of 17 variation sites were identified, denoted as SNP1, SNP2, SNP3, Indel4, Indel5, SNP6, SNP7, SNP8, SNP9, Indel10, SNP11, SNP12, SNP13, Indel14, SNP15, SNP16, and SNP17. Figure 1The 17 variant 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. These variants form two haplotypes (…). Figure 1 Among them, TaB3-2A2-Hap1 is located at positions 88982491, 88982578, 88982609, 88982782, 88982827, 88982942, 88982997, 88983009, 88983055, 88983100, 88983159, and 889832 in the Chinese spring wheat reference genome sequence RefSeq v1.0. The allelic variations at positions 93, 88983810, 88988219, 88988484, 88988516, and 88988526 are A, A, T, C, A, G, A, C, C, A, G, A, T, TA, G, T, and A, respectively; TaB3-2A2-Hap2 is found in the Chinese spring wheat reference genome sequence RefSeq. The allelic variations at positions 88982491, 88982578, 88982609, 88982782, 88982827, 88982942, 88982997, 88983009, 88983055, 88983100, 88983159, 88983293, 88983810, 88988219, 88988484, 88988516, and 88988526 in v1.0 are G, G, C, CG, AAGG, A, G, G, T, AC, A, C, C, T, C, C, and G, respectively. Seventeen variant sites in haplotypes TaB3-2A2-Hap1 and TaB3-2A2-Hap2 are linked.

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

[0069] Kompetitive allele-specific PCR (KASP) molecular markers can select for target traits at the DNA level. They offer accurate genotyping, high throughput, and low cost, making them one of the most suitable molecular marker technologies for assisted breeding. The inventors of this invention, through extensive experimentation and using Polymarker (https: / / www.polymarker.info / ), designed a primer set K-2A-SNP13 suitable for KASP identification of the wheat TaB3-2A2 haplotype at SNP13 on wheat chromosome 2A (position 88983810 in the Chinese spring wheat reference genome sequence RefSeq v1.0). The primer sequence specificity was evaluated using WheatOmics (http: / / 202.194.139.32 / ).

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

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

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

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

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

[0075] The K-2A-SNP13 marker can identify the nucleotide at position 71 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), which is either T or C, and is represented by y in SEQ ID No. 4.

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

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

[0078] Example 3: Application of molecular marker K-2A-SNP13 and its specific primer set in the identification of haplotype TaB3-2A2 in natural wheat populations

[0079] 1. Field phenotypic identification and data analysis of 138 natural wheat populations in the Huang-Huai region

[0080] 138 natural wheat varieties from the Huang-Huai wheat region were planted in Anyang, Henan and Suixi, Anhui in 2012-2013 and 2013-2014, and in Anyang, Henan and Gaoyi, Hebei in 2014-2015. A completely randomized block design with three replicates was used, with single-row plots, row length 1.5m, row width 0.2m, and 50 grains / row. Field management practices followed local wheat field management standards.

[0081] Phenotypic data for yield-related traits were provided by Li Jihu of our laboratory (Li et al., 2021), as mentioned above. Grain storage protein content was analyzed using a near-infrared reflectance spectrometer (Perten DA 7200, Springfield, IL, USA). The specific detection steps included: 1. Sample preparation: Cleaning the wheat grain samples to be tested, removing bran, damaged grains, and other impurities to avoid affecting the analytical results; 2. Sample loading: Evenly loading the wheat samples to be tested into the sample box, ensuring a smooth sample surface to avoid light scattering and reflection interference; 3. Spectral scanning: Scanning the samples using a near-infrared analyzer to collect spectral data in the near-infrared region. Each sample was typically scanned multiple times, and the average value was taken to improve data reliability. The total protein content obtained in the wheat grains is the grain storage protein content. The BLUP values ​​of the grain storage protein content and yield traits are shown in Table 1.

[0082] 2. Detection of genotypes of wheat varieties in the Huang-Huai region using the molecular marker K-2A-SNP13

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

[0084] The quality and concentration of genomic DNA must meet the requirements for PCR, with the following standards: agarose gel electrophoresis showing a single DNA band without obvious diffusion; a Nanodrop 2100 (Thermo) UV spectrophotometer showing an A260 / A280 ratio between 1.8 and 2.0 (indicating no protein contamination in the DNA sample), an A260 / A230 ratio between 1.8 and 2.0 (indicating low salt ion concentration in the DNA sample), and no obvious light absorption at 270 nm (indicating no phenol contamination in the DNA sample); the concentration of the wheat genomic DNA to be tested should be 50-200 ng / μL.

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

[0086] Using wheat genomic DNA as a template, PCR amplification was performed using the KASP primer set synthesized in Example 2 to obtain the amplification products. The reaction system consisted of: 2.0 μL KASP 2×Master Mix (LGC, catalog number: 13448166), 0.048 μL KASP primers (a mixture of 3 primers, with a total concentration of 50 μM, where the molar ratio of two forward primers to one reverse primer was 2:2:5), and 2.0 μL template DNA (50 ng / μL). The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using a touch-down program, decreasing by 0.6℃ per cycle) for 1 min, for 10 cycles; 94℃ denaturation for 20 s, 55℃ for 1 min, for another 31 cycles.

[0087] (3) Fluorescence signal acquisition and genotyping

[0088] Fluorescence signals were detected using the PHERAstar Plus autofocus fluorescence multifunction microplate reader (BMG Labtech GmbH, Ortenberg, Germany), and genotyping was performed using KlusterCaller software (LGC, Hoddesdon, UK). Fluorescence values ​​were read by scanning the FAM and HEX beams of the microplate reader after the PCR amplification products cooled to below 40℃ (FAM fluorescent tags were read at excitation wavelengths of 485nm and emission wavelengths of 520nm, and HEX fluorescent tags were read at excitation wavelengths of 528nm and emission wavelengths of 560nm). The genotype of the wheat sample based on the SNP13 locus was determined based on the fluorescence signal color. The specific determination principle is as follows: if the wheat sample shows a blue fluorescence signal based on the K-2A-SNP13 marker, the genotype of the wheat sample at the SNP13 locus is TT homozygous; if the wheat sample shows a red fluorescence signal based on the K-2A-SNP13 marker, the genotype of the wheat sample at the SNP13 locus is CC homozygous.

[0089] Based on the linkage relationships of the 17 variant sites described in Example 1, the wheat sample with genotype TT at SNP13 is haplotype TaB3-2A2-Hap1. The wheat sample with genotype CC at SNP13 is haplotype TaB3-2A2-Hap2.

[0090] The haplotype classification of 138 wheat lines is shown in Table 1. For detailed classification results, please refer to [link to table]. Figure 2 .

[0091] 3. Association analysis between wheat TaB3-2A2 genotype and storage protein content and yield

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

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

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] Note: Homozygous wheat samples with the genotype T at SNP13 are denoted as TT, and these wheat samples are haplotypes of TaB3-2A2-Hap1 (Hap1 in the table). Homozygous wheat samples with the genotype C at SNP13 are denoted as CC, and these wheat samples are haplotypes of TaB3-2A2-Hap2 (Hap2 in the table). SNP13 is a SNP site in the wheat genome, located at nucleotide 71 of SEQ ID NO. 4, and its nucleotide type is either T or C. All wheat samples tested were homozygous wheat lines.

[0101] Table 2. Phenotypic relationship analysis between TaB3-2A2 gene haplotype 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 the two genotypes TaB3-2A2-Hap1 and TaB3-2A2-Hap2. * indicates a significant difference (P<0.05).

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

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

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

[0107] The conclusion is as follows:

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

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

[0110] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of substances for detecting SNP site polymorphisms or genotypes, characterized in that, The application is at least one of the following: A1) Application in identifying or assisting in the identification of wheat grain storage protein content; A2) Application in the preparation of products for identifying or assisting in the identification of wheat grain storage protein content; A3) Application in screening or assisting in screening wheat varieties with high grain storage protein content; A4) Application in the preparation of products using wheat varieties with high grain storage protein content for screening or assisted screening; A5) Application in screening or assisting in screening wheat varieties with low grain storage protein content; A6) Application in the preparation of products for screening or assisting in the screening of wheat varieties with low grain storage protein content; A7) Application in breeding and / or assisted breeding of wheat grain storage protein content traits; A8) Application in the preparation of products for breeding and / or assisted breeding of wheat grain storage protein content traits; The SNP site is SNP13, which is an SNP site in the wheat genome, such as the 71st nucleotide of SEQ ID No. 4, and its nucleotide type is T or C. The grain storage protein content of wheat with the CC genotype at SNP13 in the wheat genome is higher or candidate higher than that of wheat with the TT genotype at SNP13 in the wheat genome.

2. The application according to claim 1, characterized in that, The substance used to detect SNP site polymorphisms or genotypes is a primer composition for amplifying wheat genomic DNA fragments, including the SNP13 site.

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

3.

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

3.

5. The use of the reagent and / or kit in at least one of B1) to B4) below, wherein the reagent and / or kit contains the primer composition of any one of claims 2 to 4; B1) Application in identifying or assisting in the identification of wheat grain storage protein content; B2) Application in screening or assisting in screening wheat varieties with high grain storage protein content; B3) Application in screening or assisting in screening wheat varieties with low grain storage protein content; B4) Application in breeding and / or assisted breeding of wheat grain storage protein content traits; The grain storage protein content of wheat with the CC genotype at SNP13 in the wheat genome is higher or candidate higher than that of wheat with the TT genotype at SNP13 in the wheat genome.

6. A method for identifying or assisting in the identification of wheat grain storage protein content, characterized in that, The method includes using a substance for detecting the polymorphism or genotype of the SNP13 site to detect the genotype of the SNP13 site in the wheat to be tested, and identifying or assisting in identifying the storage protein content of wheat grains based on the polymorphism or genotype of the SNP13 site in the wheat to be tested. The SNP13 site is a SNP site in the wheat genome, which is the 71st nucleotide of SEQ ID No. 4, and its nucleotide type is T or C; The grain storage protein content of wheat with the CC genotype at SNP13 in the wheat genome is higher or candidate higher than that of wheat with the TT genotype at SNP13 in the wheat genome.

7. The method according to claim 6, characterized in that, The method for detecting the genotype of the SNP13 site in the wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, performing PCR amplification using the primer composition described in claim 5, and obtaining PCR products. The polymorphism or genotype of the SNP13 site is determined based on the sequencing results of the PCR product or the fluorescence signal.

8. A method for wheat breeding, characterized in that, The method includes any of the following: C1) Select wheat with the genotype TT at the SNP13 locus in claim 1 as the parent for breeding, wherein the TT genotype represents a homozygous wheat genome in which the nucleotide type at the SNP13 locus is T; C2) Select wheat with the genotype CC at the SNP13 locus in claim 1 as the parent for breeding, wherein the CC genotype represents a homozygous wheat genome in which the nucleotide type at the SNP13 locus is C; The grain storage protein content of wheat with the CC genotype at SNP13 in the wheat genome is higher or candidate higher than that of wheat with the TT genotype at SNP13 in the wheat genome.

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