Wheat quality marker gli-gamma1-i and applications

Abstract

The application discloses a wheat quality marker Gli-gamma 1-I and application. The application provides application of a single nucleotide polymorphism of a SNP site on a 1D chromosome of wheat or a substance for detecting the single nucleotide polymorphism of the SNP site on the 1D chromosome of the wheat in identification or auxiliary identification of wheat quality to be tested; the SNP site is located at 511 bp downstream of TraesFLD1D01G005600 (Gli-gamma 1-1D) ATG on the 1D chromosome of the wheat; and the nucleotide at the SNP site is C or T. The application provides a molecular marker for high-quality wheat breeding, is used for screening and creating high-quality materials, enriches wheat quality breeding molecular markers, effectively improves breeding efficiency, solves the problem of low quality breeding efficiency caused by the fact that the seed amount is small and quality analysis cannot be performed in a quality breeding process, realizes early-stage molecular marker assisted selection in quality breeding, and improves breeding accuracy and purpose.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a wheat quality marker Gli-γ1-I and application thereof. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is one of the most widely cultivated crops in the world, providing abundant nutrients and proteins for human beings. Gluten imparts elasticity and extensibility to dough, which is the main determinant of wheat flour quality, determining the use of wheat flour. Wheat flour with good elasticity, i.e. strong gluten wheat flour, is suitable for making bread, while wheat flour with poor elasticity and good extensibility, i.e. weak gluten wheat flour, is suitable for making biscuits. Gluten is composed of glutenin and prolamin. Glutenin is composed of high molecular weight glutenin subunits and low molecular weight glutenin subunits, which form gluten polymers through intermolecular disulfide bonds. Prolamin affects the viscosity of dough through interaction with glutenin, and is generally considered to be closely related to dough extensibility. Prolamin is divided into four types, i.e. α / β-, γ-, ω- and δ-prolamin, according to the difference in primary structure of amino acids.

[0003] The contribution of gluten to quality is relatively clear. The genes encoding high-molecular-weight glutenin (HMW-GS) are located on the long arms of chromosomes A, B, and D of the first homoeologous group of the wheat genome and are controlled by multiple alleles. Two genes are linked and inherited on the same chromosome. In the genome of common wheat, the genes encoding HMW-GS are located at Glu-A1, Glu-B1, and Glu-D1 loci, and each locus encodes two subunits of HMW-GS, which are divided into x and y subunits according to their molecular weights. The x subunit has a larger molecular weight, and the y subunit has a smaller molecular weight. Due to gene silencing in wheat, Glu-Ay is usually in a silent state, and Glu-By is sometimes not expressed. Therefore, only four or five subunits of HMW-GS are expressed in common wheat varieties. The high-molecular-weight subunits 1Ax2*, Bx14+By15, Bx17+By18, Dx5+Dy10 have a positive effect on baking quality, while Dx2+Dy12 has a negative effect on baking quality, but when the grain protein content exceeds 15%, the effect of these factors on baking quality will weaken. Studies have shown that when the total protein content is constant, increasing the Glu / Gli ratio will increase the dough development time, peak resistance to mixing, maximum resistance to extension, and bread volume, and weaken the extensibility of the dough. When the Glu / Gli ratio is constant, increasing the total protein content can still increase the gluten strength and bread volume. Due to the large number of members in the prolamin family, 29 α-prolamins, 18 γ-prolamins, and 10 ω-prolamins were identified in the reference genome of 'Changum' which has published genome sequence. Prolamins are distributed in clusters, and almost all of them are encoded by Gli-1 and Gli-2 loci located on chromosomes 1 and 6 of the first homoeologous group. The Gli-1 locus encodes ω-prolamins and γ-prolamins, and the Gli-2 locus encodes α / β-prolamins. At the same time, prolamins are highly variable in nature, and more than 1,200 α-prolamin sequences and 400 γ-prolamin sequences have been identified from different wheat. The structure of prolamin-encoding genes is complex, with a large number of intergenic and intragenic repeat sequences, which makes it difficult to obtain DNA sequences of some prolamin-encoding genes through sanger sequencing and high-throughput short-read sequencing. There are a large number of gap regions in the prolamin-encoding positions of the Chinese Spring reference genome and the 10+ pan-genome. Therefore, the contribution of specific prolamin subunits to quality is not clear, and the application of molecular marker-assisted breeding of prolamin genes in wheat is limited.

[0004] At present, the main indicators of quality analysis are the volume of bread, dough rheological analysis, gluten index and SDS sedimentation value. Because at least 1000g of flour is needed to make bread, more than 50g of flour is needed for rheological analysis, and more than 10g of flour is needed for gluten index, the required materials are relatively more, and it takes a long time, which is not conducive to batch and rapid quality analysis. The above factors also lead to the fact that in breeding, quality analysis cannot be selected in early generations of breeding, increasing the uncertainty of breeding, resulting in a large amount of quality breeding work. The SDS sedimentation value only needs 2g of whole wheat flour at a time, and the determination is fast, which is a stable and rapid wheat quality detection indicator.

[0005] Previous studies have shown that prolamin proteins are closely related to wheat quality. For example, adding γ-prolamin protein to wheat flour can shorten the stability time and tensile resistance of the dough, resulting in a decrease in dough gluten strength. At the same time, when the clustered α-type prolamin protein coding gene is deleted, the quality of wheat flour can be improved. At present, various phenotypic changes have been obtained by reducing the expression of prolamin protein or deleting prolamin protein through RNAi and gene editing technology. By targeting the expression of ω-1, ω-2 and ω-5 coding genes through RNAi, the gluten content and gluten strength can be improved; at the same time, the expression of γ-type prolamin protein is reduced through RNAi, but no visible effect on quality is observed. By reducing α-type prolamin protein through RNAi and gene expression, the quality of some transgenic lines increases, but the quality of some transgenic lines decreases. Therefore, prolamin protein is closely related to quality, and the transgenic offspring may produce different quality phenotypic changes due to off-target problems, which requires the use of correlation analysis and other means to study the contribution of prolamin protein sequence to quality, so as to develop molecular markers to serve traditional breeding. SUMMARY

[0006] The purpose of the present application is to provide a wheat quality marker Gli-γ1-I and its application.

[0007] In a first aspect, the present application claims the use of a single nucleotide polymorphism of the following SNP site on wheat 1D chromosome or a substance for detecting the single nucleotide polymorphism of the following SNP site on wheat 1D chromosome in identifying or assisting in identifying the quality of a wheat to be tested;

[0008] The SNP site is located at 511bp downstream of the ATG of TraesFLD1D01G005600 (Gli-γ1-1D) on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16); the nucleotide at the SNP site is C or T.

[0009] Further, the substance for detecting the single nucleotide polymorphism of the SNP site on the 1D chromosome of wheat is the KASP primer described in the second aspect hereinafter or the reagent or kit described in the third aspect hereinafter.

[0010] In a second aspect, the present application claims to protect a KASP primer for identifying or assisting in identifying the quality of wheat.

[0011] The KASP primer claimed in the present application consists of primer 1, primer 2 and primer 3; the primer 1 is a single-stranded DNA from 5' end to 3' end in turn a tag sequence A and the nucleotide sequence of SEQ ID No. 1 at 22-41; the primer 2 is a single-stranded DNA from 5' end to 3' end in turn a tag sequence B and the nucleotide sequence of SEQ ID No. 2 at 22-41; the primer 3 is a single-stranded DNA with the nucleotide sequence as shown in SEQ ID No. 3 in the sequence listing.

[0012] Further, the nucleotide sequence of the tag sequence A is SEQ ID No. 1 at 1-21; the nucleotide sequence of the tag sequence B is SEQ ID No. 2 at 1-21.

[0013] Further, the primer 1 is a single-stranded DNA with the nucleotide sequence as shown in SEQ ID No. 1; the primer 2 is a single-stranded DNA with the nucleotide sequence as shown in SEQ ID No. 2.

[0014] In a third aspect, the present application claims to protect a reagent or kit for identifying or assisting in identifying the quality of wheat.

[0015] The reagent or kit for identifying or assisting in identifying the quality of wheat claimed in the present application contains the KASP primer described in the second aspect above.

[0016] Further, the reagent or kit further contains a fluorescent probe A, a fluorescent probe B, a quencher probe A and a quencher probe B.

[0017] The nucleotide sequence of the fluorescent probe A is the same as the nucleotide sequence of the tag sequence A, and a fluorescent group A is connected to the 5' end; the nucleotide sequence of the quencher probe A is reverse complementary to the nucleotide sequence of the tag sequence A, and a quencher group is connected to the 3' end.

[0018] The nucleotide sequence of the fluorescent probe B is the same as the nucleotide sequence of the tag sequence B, and a fluorescent group B is connected to the 5' end; the nucleotide sequence of the quencher probe B is reverse complementary to the nucleotide sequence of the tag sequence B, and a quencher group is connected to the 3' end.

[0019] In the specific embodiments of the present application, the fluorescent group A is HEX; the fluorescent group B is FAM; and the quencher group is BHQ.

[0020] In a fourth aspect, the present application claims the use of the KASP primer as defined in the second aspect above or the reagent or kit as defined in the third aspect above in any one of:

[0021] (A1) identifying or assisting in identifying wheat quality;

[0022] (A2) comparing the quality of wheat to be tested;

[0023] (A3) breeding wheat single plant or strain or line or variety with relatively better quality;

[0024] (A4) wheat breeding, such as high-quality wheat breeding.

[0025] In a fifth aspect, the present application claims the use of the set of proteins or the set of nucleic acid molecules in any one of:

[0026] (A1) identifying or assisting in identifying wheat quality;

[0027] (A2) comparing the quality of wheat to be tested;

[0028] (A3) breeding wheat single plant or strain or line or variety with relatively better quality;

[0029] (A4) wheat breeding, such as high-quality wheat breeding.

[0030] The set of proteins consists of the protein as shown in SEQ ID No. 4 (Gli-γ1-I protein) and the protein as shown in SEQ ID No. 5 (Gli-γ1-II protein).

[0031] The set of nucleic acid molecules consists of the DNA molecule as shown in SEQ ID No. 8 (corresponding to TraesFLD1D01G005600-I in the examples), the DNA molecule as shown in SEQ ID No. 12 (corresponding to TraesFLD1D01G005600-V in the examples), the DNA molecule as shown in SEQ ID No. 9 (corresponding to TraesFLD1D01G005600-II in the examples), the DNA molecule as shown in SEQ ID No. 10 (corresponding to TraesFLD1D01G005600-III in the examples), the DNA molecule as shown in SEQ ID No. 11 (corresponding to TraesFLD1D01G005600-IV in the examples), and the DNA molecule as shown in SEQ ID No. 13 (corresponding to TraesFLD1D01G005600-VI in the examples).

[0032] In a sixth aspect, the present application claims any one of the following methods:

[0033] Method I: a method for comparing the quality of a wheat to be tested, comprising the following steps: detecting the nucleotide at the SNP site on the 1D chromosome of the wheat to be tested, determining the genotype of the wheat to be tested, and determining the quality of the wheat to be tested according to the genotype of the wheat to be tested, wherein the quality of the wheat to be tested with a C:C genotype is higher than or is expected to be higher than the quality of the wheat to be tested with a T:T genotype.

[0034] wherein the wheat to be tested is of the Gli-γ1-I type or the Gli-γ1-II type.

[0035] Method II: a method for breeding a wheat single plant or strain or line or variety with relatively good quality, comprising the following steps: detecting the nucleotide at the SNP site on the 1D chromosome of the wheat to be tested, determining the genotype of the wheat to be tested, selecting the wheat to be tested with a C:C genotype at the SNP site on the 1D chromosome as a parent for breeding, and selecting the wheat with a C:C genotype at the SNP site on the 1D chromosome in each generation of breeding, and finally obtaining a wheat single plant or strain or line or variety with relatively good quality.

[0036] wherein the wheat to be tested is of the Gli-γ1-I type or the Gli-γ1-II type.

[0037] In each of the above methods, the SNP site is located at 511 bp downstream of the TraesFLD1D01G005600 (Gli-γ1-1D) ATG on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16); the nucleotide at the SNP site is C or T;

[0038] the C:C genotype is a homozygous type with C at 511 bp downstream of the TraesFLD1D01G005600 (Gli-γ1-1D) ATG on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16);

[0039] the T:T genotype is a homozygous type with T at 511 bp downstream of the TraesFLD1D01G005600 (Gli-γ1-1D) ATG on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16).

[0040] In addition, in theory, there is a C:T genotype in addition to the C:C genotype and the T:T genotype. The C:T genotype is a heterozygous type with C and T at 511 bp downstream of the TraesFLD1D01G005600 (Gli-γ1-1D) ATG on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16).

[0041] In the above methods, the "detection of nucleotides at the SNP site on the 1D chromosome of the wheat to be tested to determine the genotype of the wheat to be tested" is performed according to a method comprising the following steps: PCR amplification of the genomic DNA of the wheat to be tested using the reagent or kit described in the third aspect of the preceding text, fluorescence signal scanning of the amplified product, and then determining the genotype of the SNP site on the 1D chromosome of the wheat to be tested according to the following:

[0042] If the fluorescence signal of the amplification product of the wheat to be tested is the signal of the fluorescent group A, the SNP site of the wheat to be tested is the C:C genotype.

[0043] If the fluorescence signal of the amplification product of the wheat to be tested is the signal of the fluorescent group B, the SNP site of the wheat to be tested is the T:T genotype.

[0044] If the fluorescence signal of the amplification product of the wheat to be tested is the signal of the fluorescent group A and the signal of the fluorescent group B, the SNP site of the wheat to be tested is the C:T genotype.

[0045] In a seventh aspect, the present application claims protection of any one of the following methods:

[0046] Method III: A method for comparing the quality of wheat to be tested, comprising the following steps: detecting the Gli-γ1 type of the wheat to be tested, and determining the quality of the wheat to be tested according to the Gli-γ1 type of the wheat to be tested: the quality of the wheat to be tested of the Gli-γ1-I type is higher or is a candidate for being higher than the quality of the wheat to be tested of the Gli-γ1-II type.

[0047] Wherein the wheat to be tested is of the Gli-γ1-I type or the Gli-γ1-II type.

[0048] Method IV: A method for breeding wheat single plants or strains or lines or varieties with relatively good quality, comprising the following steps: detecting the Gli-γ1 type of the wheat to be tested, selecting the wheat to be tested of the Gli-γ1-I type as a parent for breeding, and selecting wheat of the Gli-γ1-I type in each generation of breeding to ultimately obtain wheat single plants or strains or lines or varieties with relatively good quality.

[0049] Wherein the wheat to be tested is of the Gli-γ1-I type or the Gli-γ1-II type.

[0050] The Gli-γ1-I type refers to containing a coding gene (homozygous) of a protein (Gli-γ1-I protein) shown in SEQ ID No. 4 on the 1D chromosome of wheat; corresponding to the C:C genotype described in the preceding text.

[0051] The Gli-γ1-II type refers to containing the coding gene (homozygous) of the protein shown as SEQ ID No. 5 (Gli-γ1-II protein) on the 1D chromosome of wheat; corresponding to the aforementioned T:T genotype.

[0052] The Gli-γ1 type includes the Gli-γ1-I type, the Gli-γ1-II type, the Gli-γ1-III type and the Gli-γ1-IV type.

[0053] The Gli-γ1-III type refers to containing the coding gene (homozygous) of the protein shown as SEQ ID No. 6 (Gli-γ1-III protein) on the 1D chromosome of wheat.

[0054] The Gli-γ1-IV type refers to containing the coding gene (homozygous) of the protein shown as SEQ ID No. 7 (Gli-γ1-IV protein) on the 1D chromosome of wheat.

[0055] Further, the coding gene of the protein shown as SEQ ID No. 4 (Gli-γ1-I protein) is shown as SEQ ID No. 8 (corresponding to TraesFLD1D01G005600-I in the examples) or SEQ ID No. 12 (corresponding to TraesFLD1D01G005600-V in the examples).

[0056] Further, the coding gene of the protein shown as SEQ ID No. 5 (Gli-γ1-II protein) is shown as SEQ ID No. 9 (corresponding to TraesFLD1D01G005600-II in the examples), SEQ ID No. 10 (corresponding to TraesFLD1D01G005600-III in the examples), SEQ ID No. 11 (corresponding to TraesFLD1D01G005600-IV in the examples) or SEQ ID No. 13 (corresponding to TraesFLD1D01G005600-VI in the examples).

[0057] Further, the coding gene of the protein shown as SEQ ID No. 6 (Gli-γ1-III protein) is shown as SEQ ID No. 14 (corresponding to TraesFLD1D01G005600-VII in the examples).

[0058] Further, the coding gene of the protein shown as SEQ ID No. 7 (Gli-γ1-IV protein) is shown as SEQ ID No. 15 (corresponding to TraesFLD1D01G005600-VIII in the examples).

[0059] In the above aspects, the genotype of the wheat is the aforementioned C:C genotype or the aforementioned T:T genotype. Alternatively, the wheat is the aforementioned Gli-γ1-I type or the aforementioned Gli-γ1-II type.

[0060] In the present application, the quality of the wheat is represented by the SDS sedimentation value. The higher the SDS sedimentation value, the better the quality of the wheat.

[0061] In the detailed embodiments of the present application, the wheat is a variety with high molecular gluten subunit of "1Ax null, Bx7+By8, Dx2+Dy12" or "1Ax, 1Bx7+1By8, 1Dx2+1Dy12". Further, the wheat is a non-1B1R ectopic system.

[0062] The present application has the following beneficial effects: providing a molecular marker for high-quality wheat breeding, for screening and creating high-quality materials; at the same time, enriching the molecular markers for wheat quality breeding, effectively improving the breeding efficiency, solving the problem of low efficiency of quality breeding due to the small amount of seeds and the inability to perform quality analysis during the quality breeding process, realizing early-stage molecular marker assisted selection for quality breeding, and improving the breeding accuracy and purpose. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 TraesFLD1D01G005600 gene editing. a is TraesFLD1D01G005600 gene editing. The gene has a base insertion at the sgRNA position, introducing an amino acid frameshift mutation, causing loss of gene function. b is TraesFLD1D01G005600 (Gli-γ1-1D) encoding Gli-γ1. The change in prolamin was identified by A-PAGE electrophoresis technology, and it was found that the Gli-γ1 band was missing in the TraesFLD1D01G005600 mutant line, and it was considered that Gli-γ1 was mainly encoded by TraesFLD1D01G005600.

[0064] Figure 2 TraesFLD1D01G005600 haplotype.

[0065] Figure 3 Four kinds of amino acid sequence differences of Gli-γ1. a is amino acid sequence alignment. b is sequence difference position.

[0066] Figure 4 Distribution of Gli-γ1-I and Gli-γ1-II in traditional varieties, breeding varieties, and high-quality varieties.

[0067] Figure 5Quality analysis of Gli-γ1-I and Gli-γ1-II. High molecular glutenin subunit background: 1A-null, 1Bx7+1By8, 1Dx2+1Dy12.

[0068] Figure 6 Molecular marker development for Gli-γ1-I. a KASP marker position. KASP primers were designed using a 511 bp SNP to differentiate Gli-γ1-I and Gli-γ1-II. b KASP marker detection. The developed marker was able to efficiently differentiate the two haplotypes using 40 materials carrying Gli-γ1-I and 40 materials carrying Gli-γ1-II for validation. c RIL population constructed from Zang 1817 and Nongda 3331 to validate the reliability of the marker. Nongda 3331 carries Gli-γ1-I and Zang 1817 carries Gli-γ1-II, and the offspring using non-1B1R, high molecular glutenin subunit background of 1Ax, 1Bx7+1By8, 1Dx2+1Dy12 for validation. DETAILED DESCRIPTION

[0069] The application will be further described in conjunction with the preferred embodiments thereof with reference to the following examples. The following examples are presented by way of illustration of the application and therefore should not be construed to limit the scope of the present application. The following examples provided herein serve as a guide for further improvement by those having ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0070] The experimental methods used in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0071] 1. DNA extraction

[0072] DNA was extracted using the CTAB method. The specific operation is as follows:

[0073] (1) Selecting young leaves at the seedling stage and placing them in a round-bottom 2.0 mL centrifuge tube with steel balls.

[0074] (2) Freezing the sample tube in liquid nitrogen until the liquid nitrogen boils off.

[0075] (3) Placing the frozen sample tube in a grinding box equipped with a grinder (the grinding box is pre-cooled in liquid nitrogen) and grinding.

[0076] (4) Adding 600 μL of 2x CTAB (SL2071-500 mL, Coolaber, Beijing, China) to the ground sample and incubating at 60°C for 40 min, inverting and mixing every 10 min during the incubation.

[0077] (5) Add 600 μL chloroform in the fume hood, invert to mix, stand for 10 min, centrifuge at 12000 rpm, room temperature for 10 min.

[0078] (6) Take 600 μL supernatant to a new 1.5 mL centrifuge tube, add 600 μL isopropanol in the centrifuge tube, invert to mix, stand for precipitation at -20 °C overnight.

[0079] (7) Centrifuge at 12000 rpm, 4 °C for 5 min.

[0080] (8) Add 1 mL 75% ethanol, centrifuge at 12000 rpm, 4 °C for 5 min, pour off the supernatant, wash the DNA, repeat once.

[0081] (9) Blow dry the DNA overnight.

[0082] (10) Add 100 μL ddH2O to dissolve the DNA.

[0083] 2. SDS analysis

[0084] According to the method introduced by Ma Chuanxi et al., the trace SDS sedimentation value of 2 g of flour is determined, and the specific steps are as follows:

[0085] Reagent preparation:

[0086] Bromophenol blue stock solution (1 L): 1 g of bromophenol blue is dissolved in 1 L of deionized water.

[0087] Bromophenol blue working solution (1 L): 10 ml of bromophenol blue stock solution + 990 ml of deionized water.

[0088] Lactic acid aqueous solution: 100 ml of 85% lactic acid (Shanghai test, 30108518) is added with 800 ml of water, and refluxed for more than 6 h.

[0089] 10% SDS (1 L): 400 ml of deionized water + 100 g of SDS, mix well, dissolve at 60 °C, and make up to 1 L.

[0090] 1:50 lactic acid-SDS working solution (1 L): 20 ml of lactic acid aqueous solution + 200 ml of 10% SDS + 800 ml of deionized water.

[0091] (1) Weigh 2 g ± 0.01 g of flour sample in a 35 mL cylinder with a stopper (three times);

[0092] (2) Add 16.7 mL of bromophenol blue working solution, and add a stopper. Mix the flour and solution well, and put it on the sedimentation value tester (frequency 13) for 5 min;

[0093] (3) Remove the cylinder, add 16.7 mL of 1 :50 lactic acid-SDS working solution, cover the plug, mix up and down, place the cylinder on the shaker (frequency 13) and shake for 5 min;

[0094] (4) Remove the cylinder from the shaker, immediately stand it on the horizontal table, stand for 5 min, quickly read the volume of the precipitate, which is the micro-SDS-sedimentation value, and the reading is estimated to 0.1 mL.

[0095] 3. KASP marker development

[0096] The 511 bp base difference downstream of the ATG of the Gli-γ1-1D gene was used to develop a KASP marker for distinguishing Gli-γ1-I and Gli-γ1-II.

[0097] A 8 μL reaction system was established using a 96-well PCR plate: DNA template 50 ng; HiGeno 2x Probe Mix A 4 μL; primer 1 μL (0.12 μM upstream primer F1, 0.12 μM upstream primer F2, 30 μM downstream primer R); ddH2O to 8 μL.

[0098] Amplification was performed by touchdown PCR program: 95°C for 10 min; 95°C for 20 sec, 65-55°C for each cycle, reducing 0.6°C, for a total of 10 cycles; 95°C for 20 sec, 55°C for 40 sec, 32 cycles.

[0099] The fluorescence signal was read by CFX96 real-time quantitative PCR instrument (Bio-Rad, Hercules, California, USA).

[0100] The fluorescence signal is converted into genotype by BIO-RAD CFX Maestro. According to the relative fluorescence value, the samples are clustered into clusters, and the genotype of the SNP site on the 1D chromosome of the wheat to be tested in the application (i.e., whether the base at 511 bp downstream of the ATG of TraesFLD1D01G005600 (Gli-γ1-1D) on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16) is C or T) is determined according to the sample cluster and the fluorescence type: if the fluorescence signal data of the amplification product of the wheat to be tested is close to the Y axis (HEX signal) after KlusterCaller analysis, the genotype of the SNP site on the 1D chromosome of the wheat to be tested in the application is C:C (i.e., the base at 511 bp downstream of the ATG of TraesFLD1D01G005600 (Gli-γ1-1D) on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16) is C:C homozygous); if the fluorescence signal data of the amplification product of the wheat to be tested is close to the X axis (FAM signal) after KlusterCaller analysis, the genotype of the SNP site on the 1D chromosome of the wheat to be tested in the application is T:T (i.e., the base at 511 bp downstream of the ATG of TraesFLD1D01G005600 (Gli-γ1-1D) on the 1D chromosome of wheat (corresponding to position 20 of SEQ ID No. 16) is T:T homozygous).

[0101] Example 1, Wheat quality marker Gli-γ1-I and application

[0102] I. TraesFLD1D01G005600 (Gli-γ1-1D) encodes Gli-γ1

[0103] By comparing the prolamin sequences, specific sgRNAs were designed and delivered into the wheat recipient Fielder. After screening, we obtained a material in which TraesFLD1D01G005600 (Gli-γ1-1D) had a base insertion, and other γ-type prolamin-encoding genes were not changed. Figure 1 A-PAGE electrophoresis found that the Gli-γ1 band spectrum in the material disappeared, and the rest of the prolamin band spectrum had no obvious change. Figure 1 This indicates that TraesFLD1D01G005600 (Gli-γ1-1D) encodes Gli-γ1.

[0104] II. Gli-γ1-I is an excellent allelic variation and can be used for quality breeding

[0105] Eight DNA sequences were obtained from 181 natural populations including improved varieties and landraces, and were named as TraesFLD1D01G005600-I (SEQ ID No. 8), TraesFLD1D01G005600-II (SEQ ID No. 9), TraesFLD1D01G005600-III (SEQ ID No. 10), TraesFLD1D01G005600-IV (SEQ ID No. 11), TraesFLD1D01G005600-V (SEQ ID No. 12), TraesFLD1D01G005600-VI (SEQ ID No. 13), TraesFLD1D01G005600-VII (SEQ ID No. 14), and TraesFLD1D01G005600-VIII (SEQ ID No. 15). Due to the wobble of the third codon during transcription, the eight DNA sequences encode four amino acid sequences. TraesFLD1D01G005600-I and TraesFLD1D01G005600-V encode Gli-γ1-I (SEQ ID No. 4), TraesFLD1D01G005600-II, TraesFLD1D01G005600-III, TraesFLD1D01G005600-IV, and TraesFLD1D01G005600-VI encode Gli-γ1-II (SEQ ID No. 5), TraesFLD1D01G005600-VII encodes Gli-γ1-III (SEQ ID No. 6), and TraesFLD1D01G005600-VIII encodes Gli-γ1-IV (SEQ ID No. 7). Figure 2 ) Sequence analysis found that the four amino acids mainly exist sequence differences in the repeat region and polyQ domain of Gli-γ1( Figure 3 )

[0106] In the 181 sequencing materials, we found that only one material was Gli-γ1-III, and two materials were Gli-γ1-IV. It is shown that Gli-γ1-III and Gli-γ1-IV are not the main types of Gli-γ1 in the 181 materials. Further research found that 113 materials were Gli-γ1-I, accounting for 62.4% of the total test materials, and 65 materials were Gli-γ1-II, accounting for 35.9% of the total sequencing materials. It is shown that Gli-γ1-I and Gli-γ1-II are the main existing forms (Table 1 and Table 2).

[0107] Table 1, haplotypes of 181 natural wheat populations

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] Table 2, sequence information of TraesFLD1D01G005600

[0114]

[0115]

[0116] We found that the proportion of Gli-γ1-I was higher than Gli-γ1-II in improved varieties, and it was speculated that the genotype was selected in the breeding process, which may be an excellent haplotype for quality Figure 4 ). Because the high molecular weight glutenin subunit plays an important role in quality, especially the composition of different high molecular weight glutenin subunits, which can have an important impact on baking quality. For example, Dx5 and Dy10 are high-quality subunits, which have a positive effect on baking quality; Dx2 and Dy12 are non-quality subunits, which have a negative effect on baking quality. Therefore, in order to exclude the difference in the contribution of different high molecular weight glutenin subunits to quality, we used 1Ax null, Bx7+By8, Dx2+Dy12 high molecular weight glutenin subunit background materials to analyze the quality of Gli-γ1-I and Gli-γ1-II. At the same time, because the 1B1R ectopic system seriously affects the quality, we selected non-1B1R ectopic system for analysis. It was found that the SDS sedimentation value of Gli-γ1-I was significantly higher than that of Gli-γ1-II (P<0.05), indicating that Gli-γ1-I is an excellent haplotype Figure 5 and Table 3).

[0117] Table 3, SDS sedimentation value of Gli-γ1-I is significantly higher than that of Gli-γ1-II in natural population

[0118]

[0119]

[0120] Note: DX2 in the table represents Dx2+Dy12, which is linked with Dx2 and Dy12.

[0121] We found a single nucleotide polymorphism at 511bp by analyzing the DNA sequences of Gli-γ1-I and Gli-γ1-II. Based on this, we developed a set of KASP molecular markers for distinguishing Gli-γ1-I and Gli-γ1-II Figure 6 in the middle a).

[0122] KASP upstream primer F1: 5'-GAAGGTGACCAAGTTCATGCTATTCCCCCAACAACAACGG -3' (SEQ ID No. 1, the underlined part is a specific fluorescent tag sequence HEX). C

[0123] KASP upstream primer F2: 5'-GAAGGTCGGAGTCAACGGATTATTCCCCCAACAACAACGG -3' (SEQ ID No. 2, the underlined part is a specific fluorescent tag sequence FAM). T

[0124] KASP downstream primer R: 5'-ATTATTGACCAGAGGGATGACACC-3' (SEQ ID No. 3).

[0125] The last base pair at the 3' end of the two upstream primers corresponds to the SNP site. The SNP site corresponds to the 20th position of SEQ ID No. 16 on the wheat 1D chromosome; the nucleotide at the SNP site is C or T (represented by Y in SEQ ID No. 16).

[0126] The upstream primer F1 is used for amplification when the nucleotide at the SNP site on the wheat 1D chromosome is C, and the upstream primer F2 is used for amplification when the nucleotide at the SNP site on the wheat 1D chromosome is T; the downstream primer R is a universal primer.

[0127] The single-stranded DNA molecule shown in SEQ ID No. 1 above and the single-stranded DNA molecule shown in SEQ ID No. 3 amplify the fragment of the C:C homozygote (i.e., the base at the 20th position of SEQ ID No. 16 on the wheat 1D chromosome is C:C homozygote) at the SNP site on the wheat 1D chromosome. The sequence of the theoretical amplification product (without specific fluorescent tag sequence) is shown in SEQ ID No. 16 (the 20th position is C).

[0128] ​​The single-stranded DNA molecule shown in SEQ ID No. 2 and the single-stranded DNA molecule shown in SEQ ID No. 3 amplify the fragment of the SNP site on the 1D chromosome of wheat, in which the nucleotide is T:T homozygous (i.e., the base at position 20 of SEQ ID No. 16 on the 1D chromosome of wheat is T:T homozygous). The sequence of the theoretical amplification product (without specific fluorescent tag sequence) is shown in SEQ ID No. 16 (position 20 is T).

[0129] The single-stranded DNA molecule shown in SEQ ID No. 1, the single-stranded DNA molecule shown in SEQ ID No. 2 and the single-stranded DNA molecule shown in SEQ ID No. 3 amplify the fragment of the SNP site on the 1D chromosome of wheat, in which the nucleotide is C:T heterozygous (i.e., the base at position 20 of SEQ ID No. 16 on the 1D chromosome of wheat is C:T heterozygous). The theoretical amplification product (without specific fluorescent tag sequence) has two strands, which are DNA fragment 1 shown in SEQ ID No. 16 (position 20 is C) and DNA fragment 2 shown in SEQ ID No. 16 (position 20 is T).

[0130] To verify the accuracy of the KASP marker, we randomly selected 40 Gli-γ1-I (homozygous) and 40 Gli-γ1-II (homozygous) materials for KASP marker typing verification, and found that the KASP typing was consistent with the sequencing results, indicating that the marker could distinguish Gli-γ1-I and Gli-γ1-II (Table 4). Figure 6 To verify the accuracy of the marker, we verified the marker in the RIL population. By KASP marker typing on the offspring of Nongda 3331 (carrying Gli-γ1-I) and Zang 1817 (carrying Gli-γ1-II) RIL offspring with high molecular weight glutenin subunit 1Ax, 1Bx7+1By8, 1Dx2+1Dy12, and quality analysis, we found that the SDS sedimentation value of Gli-γ1-I was significantly higher than that of Gli-γ1-II (Table 5), which verified that Gli-γ1-I was an excellent haplotype, and the marker was stable and reliable. Therefore, KASP of Gli-γ1-I can be used for molecular marker assisted selection in quality breeding. Figure 6

[0131] Table 4, KASP marker typing results of 40 Gli-γ1-I and 40 Gli-γ1-II materials

[0132]

[0133]

[0134] ​

[0135] Table 5. SDS sedimentation value detection results of RIL offspring of Nongda 3331 and Zang 1817

[0136]

[0137]

[0138] Note: Both parents Nongda 3331 and Zang 1817 have high gluten subunit 1D chromosome, which is 1Dx2+1Dy12 subunit, so the offspring must be 1Dx2+1Dy12 subunit.

[0139] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In general, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art beyond the scope disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of a single nucleotide polymorphism at a SNP locus on wheat 1D chromosome as follows or a substance for detecting a single nucleotide polymorphism at a SNP locus on wheat 1D chromosome as follows in identifying or assisting in identifying a wheat quality to be tested; the SNP locus is located on the 20th position of SEQ ID No. 16 on wheat 1D chromosome; the nucleotide at the SNP locus is C or T; the wheat quality is embodied by SDS sedimentation value.

2. The substance for detecting a single nucleotide polymorphism at a SNP locus on wheat 1D chromosome as follows is a KASP primer or a reagent or kit containing the KASP primer; 3. The KASP primer consists of primer 1, primer 2 and primer 3; the primer 1 is a single-stranded DNA from 5' end to 3' end in turn a tag sequence A and the 22nd-41st position of SEQ ID No. 1; the primer 2 is a single-stranded DNA from 5' end to 3' end in turn a tag sequence B and the 22nd-41st position of SEQ ID No. 2; the primer 3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No. 3 in the sequence listing; 2. Use according to claim 1, characterized in that:

4. The nucleotide sequence of the tag sequence A is the 1st-21st position of SEQ ID No. 1; the nucleotide sequence of the tag sequence B is the 1st-21st position of SEQ ID No.

2.

5. The primer 1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No. 1; the primer 2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.

2.

6. The reagent or kit further contains a fluorescent probe A, a fluorescent probe B, a quencher probe A and a quencher probe B; 3. Use according to claim 2, characterized in that:

7. The nucleotide sequence of the fluorescent probe A is consistent with the nucleotide sequence of the tag sequence A, and the 5' end is connected with a fluorescent group A; the nucleotide sequence of the quencher probe A is reverse complementary to the nucleotide sequence of the tag sequence A, and the 3' end is connected with a quencher group; 4. Use according to claim 2 or 3, characterized in that:

8. The nucleotide sequence of the fluorescent probe B is consistent with the nucleotide sequence of the tag sequence B, and the 5' end is connected with a fluorescent group B; the nucleotide sequence of the quencher probe B is reverse complementary to the nucleotide sequence of the tag sequence B, and the 3' end is connected with a quencher group.

9. The KASP primer consists of primer 1, primer 2 and primer 3; the primer 1 is a single-stranded DNA from 5' end to 3' end in turn a tag sequence A and the 22nd-41st position of SEQ ID No. 1; the primer 2 is a single-stranded DNA from 5' end to 3' end in turn a tag sequence B and the 22nd-41st position of SEQ ID No. 2; the primer 3 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No. 3 in the sequence listing; the nucleotide sequence of the tag sequence A is the 1st-21st position of SEQ ID No. 1; the nucleotide sequence of the tag sequence B is the 1st-21st position of SEQ ID No.

2.

10. The wheat quality is embodied by SDS sedimentation value.

5. A KASP primer for use in identifying or assisting in the identification of wheat quality characterised in that:

11. The primer 1 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No. 1; the primer 2 is a single-stranded DNA with a nucleotide sequence as shown in SEQ ID No.

2. ​ 6. The KASP primer according to claim 5, wherein: ​ 7. A reagent or kit for identifying or aiding in the identification of wheat quality, characterised in that: The KASP primer of claim 5 or 6 or the reagent or kit of claim 7 or 8 is used in any one of the following: (A1) identifying or assisting in identifying the quality of wheat; 8. The reagent or kit according to claim 7, wherein: (A2) comparing the quality of wheat to be tested; (A3) breeding wheat single plant or strain or line or variety with relatively better quality; (A4) wheat breeding; The quality of wheat is reflected by SDS sedimentation value. The reagent or kit further comprises fluorescent probe A, fluorescent probe B, quencher probe A and quencher probe B; The nucleotide sequence of the fluorescent probe A is identical to that of the tag sequence A, and a fluorescent group A is connected to the 5' end; the nucleotide sequence of the quencher probe A is reverse complementary to that of the tag sequence A, and a quencher group is connected to the 3' end; The nucleotide sequence of the fluorescent probe B is identical to that of the tag sequence B, and a fluorescent group B is connected to the 5' end; the nucleotide sequence of the quencher probe B is reverse complementary to that of the tag sequence B, and a quencher group is connected to the 3' end.

9. The KASP primer of claim 5 or 6 or the reagent or kit of claim 7 or 8 is used in any one of the following: (A1) identifying or assisting in identifying the quality of wheat; (A2) comparing the quality of wheat to be tested; (A3) breeding wheat single plant or strain or line or variety with relatively better quality; (A4) wheat breeding; The quality of wheat is reflected by SDS sedimentation value.

10. The set of proteins or set of nucleic acid molecules is used in any one of the following: (A1) identifying or assisting in identifying the quality of wheat; (A2) comparing the quality of wheat to be tested; (A3) breeding wheat single plant or strain or line or variety with relatively better quality; (A4) wheat breeding; The quality of wheat is reflected by SDS sedimentation value; The set of proteins consists of the protein shown as SEQ ID No. 4 and the protein shown as SEQ ID No. 5; The set of nucleic acid molecules consists of the DNA molecule shown as SEQ ID No. 8, the DNA molecule shown as SEQ ID No. 12, the DNA molecule shown as SEQ ID No. 9, the DNA molecule shown as SEQ ID No. 10, the DNA molecule shown as SEQ ID No. 11 and the DNA molecule shown as SEQ ID No.

13.

11. Any one of the following methods: Method I: a method for comparing the quality of wheat to be tested, comprising the following steps: detecting the nucleotide at the SNP site on the 1D chromosome of the wheat to be tested, determining the genotype of the wheat to be tested, and determining the quality of the wheat to be tested according to the genotype of the wheat to be tested as follows: the quality of the wheat to be tested with C:C genotype is higher than or is a candidate for being higher than that of the wheat to be tested with T:T genotype; Method II: a method for breeding wheat single plant or strain or line or variety with relatively better quality, comprising the following steps: detecting the nucleotide at the SNP site on the 1D chromosome of the wheat to be tested, determining the genotype of the wheat to be tested, selecting the wheat to be tested with C:C genotype at the SNP site on the 1D chromosome as the parent for breeding, and selecting the wheat with C:C genotype at the SNP site on the 1D chromosome in each generation of breeding, and finally obtaining wheat single plant or strain or line or variety with relatively better quality. The SNP site is located at the 20th position of SEQ ID No. 16 on the 1D chromosome of wheat; the nucleotide at the SNP site is C or T; The C:C genotype is a homozygous type of C at the 20th position of SEQ ID No. 16 on the 1D chromosome of wheat; The T:T genotype is a homozygous type of T at the 20th position of SEQ ID No. 16 on the 1D chromosome of wheat; The wheat quality is reflected by the SDS sedimentation value.

12. The method of claim 11, wherein: The "determination of the genotype of the wheat to be tested at the SNP site on the 1D chromosome of the wheat to be tested" is performed according to a method comprising the following steps: PCR amplification of the genomic DNA of the wheat to be tested by using the reagent or kit of claim 8, fluorescence signal scanning of the amplified product, and then determination of the genotype of the SNP site on the 1D chromosome of the wheat to be tested according to the following: If the fluorescence signal of the amplified product of the wheat to be tested is the signal of the fluorescent group A, then the SNP site of the wheat to be tested is the C:C genotype; If the fluorescence signal of the amplified product of the wheat to be tested is the signal of the fluorescent group B, then the SNP site of the wheat to be tested is the T:T genotype.

13. Any one of the following methods: Method III: a method for comparing the quality of wheat to be tested, comprising the following steps: detecting the Gli-γ1 type of the wheat to be tested, and determining the quality of the wheat to be tested according to the Gli-γ1 type of the wheat to be tested: the quality of the wheat to be tested of the Gli-γ1-I type is higher than or is a candidate for being higher than the quality of the wheat to be tested of the Gli-γ1-II type; Method IV: a method for breeding a wheat single plant or strain or line or variety with relatively good quality, comprising the following steps: detecting the Gli-γ1 type of the wheat to be tested, selecting the wheat to be tested of the Gli-γ1-I type as a parent for breeding, and selecting the wheat of the Gli-γ1-I type in each generation of breeding to finally obtain a wheat single plant or strain or line or variety with relatively good quality; The Gli-γ1-I type refers to containing the coding gene of the protein shown in SEQ ID No. 4 on the 1D chromosome of wheat; The Gli-γ1-II type refers to containing the coding gene of the protein shown in SEQ ID No. 5 on the 1D chromosome of wheat; The wheat quality is reflected by the SDS sedimentation value.

14. The method of claim 13, wherein: The wheat to be tested is of the Gli-γ1-I type or the Gli-γ1-II type.

15. The method of claim 13, wherein: The coding gene of the protein shown in SEQ ID No. 4 is shown in SEQ ID No. 8 or SEQ ID No.

12.

16. The method of claim 13, wherein: The coding gene of the protein shown in SEQ ID No. 5 is shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 13.

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