Application and Creation Method of Superior Allelic Variation at Gli-A2p-null Site in Bread Wheat

Abstract

This application discloses the application and creation method of bread wheat with superior allelic variation at the Gli-A2p-null locus, belonging to the field of bio-breeding technology. The technical problem this application aims to solve is how to improve the quality of processed wheat grains. To this end, this application provides products made from raw materials including wheat, wherein the wheat does not contain the gliadin genes α / β3, α / β4, and α / β5. The wheat can be wheat GA2pN, with the CGMCC accession number CGMCC No. 45699. This application, through the determination of flour protein characteristics, found that GA2pN significantly improves many quality parameters, such as sedimentation index, wet gluten content, and gluten index, which are significantly superior to wild type. This application achieves the goal of significantly improving the quality of wheat dough and bread baking quality, and has important practical significance and application value, with broad application prospects.

Description

Technical Field

[0001] This application belongs to the field of biological breeding technology, specifically relating to the application and creation method of bread wheat with superior allelic variation at the Gli-A2p-null site. Background Technology

[0002] Wheat is one of the world's most important food crops, with approximately 40% of the world's population relying on wheat flour products as their staple food. Therefore, basic and genetic research on wheat quality, yield, and other traits is of great importance to governments and researchers worldwide. As one of the world's three major grains, wheat provides 20% of the world's protein and energy, and increasing wheat production is crucial for stabilizing global food security. my country ranks first in the world in terms of wheat planting area, total production, and consumption.

[0003] Wheat is an important food crop, and a wide variety of wheat products are popular. Wheat gluten is fundamental to the quality of wheat processing; the stretchability of dough is due to the presence of gluten. The unique gluten proteins in flour are one of the main factors determining the quality characteristics of dough, influencing its kneading and stability properties, and further determining the baking properties of bread (Payne and Corfield et al., 1979). Gluten proteins are mainly composed of glutenin and gliadin. The types and amounts of gluten proteins determine the final use of wheat flour. Gluten proteins are divided into glutenin and gliadin. Glutenin is found on the first homolog of wheat. The genes encoding ω, γ, and α / β gliadin are located at the Gli-1 site on the short arm of the first homolog of wheat, and are tightly linked to low molecular weight glutenin. Glutenin is divided into high molecular weight glutenin and low molecular weight glutenin, determining the strength and elasticity of gluten and dough (Anjum et al., 2008). Allelic variations and composition of high molecular weight glutenin subunits directly affect bread baking quality and have therefore been extensively studied. Gliadins are classified into ω, γ, and α / β gliadins based on differences in electrophoretic migration rates, and they influence gluten extensibility through non-covalent bonds and interactions with glutenins. Although gliadins account for more than 50% of gluten proteins, their role in dough processing remains less clearly defined than that of high molecular weight glutenin subunits. Gliadins are encoded by multiple gene families and inherited in clusters, making their contribution to dough quality difficult to investigate (Nadeem et al., 2016).

[0004] The genes encoding α / β prolactin are located at three Gli-2 loci on the short arm of the sixth homology group in common wheat (Triticum aestivum L., AABBDD, 2n=6x=42). Each locus contains numerous α / β prolactin genes with high sequence similarity. α / β prolactin accounts for approximately 50% to 60% of the total prolactin content, making it the most abundant and highly variable type of gluten protein (Halstead-Nussloch et al., 2021). The HLA-DQ2.5 restriction epitopes Glia-α1a (PFPQPQLPY), Glia-α1b (PYPQPQLPY), Glia-α2 (PQPQLPYPQ), Glia-α3 (FRPQQPYPQ), and HLA-DQ8, along with the restriction epitopes Glia-α1 (QGSFQPSQQ) and HLA-DQ8.5Glia-α1 (QGSFQPSQQ), become potent stimulants after deamidation, activating specific CD4+ T cells and inducing a strong adaptive immune response. These are the main allergenic proteins that induce celiac disease (Pisapia et al., 2016).

[0005] Different protein components perform different functions during dough processing, ultimately determining the quality of the dough. Using mutants and gene silencing techniques can rapidly reveal the function of unknown genes and is an effective method for studying the function of wheat gluten proteins, such as chemical mutagenesis (e.g., EMS) and gene silencing techniques (e.g., RNAi) (Fu et al., 2007). However, due to the high copy number, high similarity, and highly repetitive sequences of gliadin genes, chemical mutagenesis is unlikely to achieve simultaneous silencing of multiple genes at the same site. In wheat, RNAi technology reduces gliadin content, affecting dough extensibility and leading to a reduction in bread volume. The reduction in gliadin significantly decreases celiac disease antigen peptides and significantly increases lysine content in wheat flour, enhancing its nutritional quality (Zhao et al., 2004). In protein synthesis with limited energy, the amount of synthesized protein is inversely proportional to the amount of protein synthesized; if some gliadin is lost, more amino acids will be used to synthesize glutenin. A significant reduction in α / β prolamins leads to a compensatory effect of increased levels of other gluten proteins. The dough showed no significant change compared to the control, but gluten resistance increased and extensibility slightly decreased (Becker et al., 2012). A substantial reduction in prolamins content may cause a significant decline in dough processing quality, such as reduced bread volume (Mickowska et al., 2016). Furthermore, recently developed genome editing technologies, such as the CRISPR / Cas9 system, also face numerous challenges in knocking out high-copy-count genes.

[0006] The above mutation methods are all insufficient to create effective gliadin locus deletion mutants suitable for production and application in my country. Therefore, in order to study the function of gliadin at a specific locus, it is of great practical significance to target the reduction of gliadin content and its impact on dough quality. Summary of the Invention

[0007] The technical problem to be solved by this application is: how to improve the quality of wheat grain processed products, such as how to improve the quality of wheat flour and / or the quality of bread made from said flour.

[0008] To solve the above-mentioned technical problems, this application provides a processed article, which is a product made from raw materials including wheat (the raw materials for preparing the processed article include wheat), wherein the wheat does not contain gliadin genes, and the gliadin genes are α / β3 genes, α / β4 genes and α / β5 genes.

[0009] The α / β3 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 1, the α / β4 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 2, and the α / β5 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 3.

[0010] Furthermore, in the processed product, the wheat may be wheat (Triticum sp.) GA2pN, whose preservation number at the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No. 45699.

[0011] Furthermore, the processed articles may be selected from flour, wheat starch, food and / or fermented products.

[0012] In this application, the processed product is not considered a plant under patent law. The processed product has no reproductive capacity, nor does it possess the ability to synthesize carbohydrates and proteins from inorganic substances such as water, carbon dioxide, and inorganic salts through photosynthesis to sustain its life.

[0013] In this application, the processed product may be wheat flour (such as bread flour, dumpling flour, biscuit flour, noodle flour, cake flour, granular flour, fortified flour, self-rising steamed bun flour, frying flour, pre-mixed flour, etc.), wheat starch, food (such as food made by heat processing or cold processing, food made by steaming or baking, such as bread, steamed buns, biscuits, noodles, etc.), fermented processed products, including beverages, alcoholic beverages, such as beer, liquor, alcohol, such as biofuels, etc.

[0014] Compared with ordinary wheat Xiaoyan 81, the wheat grains have one or more of the following characteristics: a1) increased glutenin content; a2) increased high molecular weight glutenin content; a3) increased low molecular weight glutenin content; a4) decreased α / β prolactin content in the grains; a5) decreased total prolactin content in the grains.

[0015] The flour is made from wheat (Triticum sp.) GA2pN grains, and compared with flour made from common wheat Xiaoyan 81 grains, it has at least one of the following characteristics: b1) increased flour sedimentation value; b2) increased wet gluten content of flour; b3) increased flour gluten index;

[0016] The food product may be bread. The bread may be bread made primarily from flour made from wheat (Triticum sp.) GA2pN grains, which has improved quality compared to bread made primarily from flour made from common wheat (Triticum sp.) 81 grains. The bread quality is selected from at least one of the following: volume, shape, crust texture, crust color, bread filling color, smoothness, texture, elasticity, taste, and aroma.

[0017] This application also provides the use of the above-mentioned wheat in the preparation of processed products.

[0018] The processed article may be at least one of the processed articles described above.

[0019] This application also provides the application of the aforementioned wheat in wheat breeding.

[0020] The breeding indicators for wheat breeding include at least one of the following: glutenin content in grains, α-gliadin content in grains, total gliadin content in grains, medium- and high molecular weight glutenin content in grains, low molecular weight glutenin content in grains, sedimentation value of flour made from wheat grains, wet gluten content of flour made from wheat grains, gluten index of flour made from wheat grains, bread volume made from flour made from wheat grains, and quality of bread made from flour made from wheat grains.

[0021] The breeding objectives of the wheat breeding program include developing wheat with at least one of the following traits:

[0022] Wheat with high glutenin content (higher than its parent).

[0023] Wheat with high glutenin content (higher than its parent).

[0024] Wheat with low α / β prolysin content in its grains (lower than the parental variety),

[0025] Wheat with low total prolamin content in its grains (lower than the parent variety),

[0026] Wheat whose flour made from wheat grains has an improved sedimentation value (higher than that of the parent wheat),

[0027] Wheat whose flour, made from wheat grains, has a higher wet gluten content (than the parent wheat).

[0028] Flour made from wheat grains has a higher gluten index (than that of the parent wheat).

[0029] Bread made from wheat grains produces bread with a higher volume (than the parent material).

[0030] Bread made from flour derived from wheat grains has improved quality (better than the parent wheat).

[0031] The bread quality is selected from at least one of the following: volume, shape, crust texture, crust color, bread filling color, smoothness, texture, elasticity, taste, and aroma.

[0032] This application also provides the application of the above-mentioned gliadin gene or substances regulating the expression of the gliadin gene in regulating wheat grain traits, wherein the wheat grain traits include any one or more of the following:

[0033] A1) Glutelin content in grains;

[0034] A2) High molecular weight glutenin content in grains;

[0035] A3), Low molecular weight glutenin content in grains;

[0036] A4) Sedimentation value of flour prepared from grains;

[0037] A5), the wet gluten content of flour prepared from grains;

[0038] A6), gluten index of flour prepared from grains;

[0039] A7) The volume of bread made from flour prepared from seeds;

[0040] A8) The quality of bread made from flour prepared from seeds;

[0041] A9), α / β prolysin content in grains;

[0042] A10), total alcohol-soluble protein content in grains.

[0043] This application also provides primers for detecting the above-mentioned prolysin gene.

[0044] In this application, the primers include primer pairs for detecting the α / β3 gene, α / β4 gene, and α / β5 gene.

[0045] In this application, the primer pair for detecting the α / β3 gene is α / β3-F and α / β3-R, wherein α / β3-F is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No. 4, and α / β3-R is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No. 5.

[0046] In this application, the primer pair for detecting the α / β4 gene is α / β4-F and α / β4-R, wherein α / β4-F is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No. 6, and α / β4-R is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No. 7.

[0047] In this application, the primer pair for detecting the α / β5 gene is α / β5-F and α / β5-R, wherein α / β5-F is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No. 8, and α / β5-R is a single-stranded DNA molecule with the nucleotide sequence of SEQ ID No. 9.

[0048] In this application, whether the processed quality-improved wheat contains the prolactin gene can be determined by primer detection of the prolactin gene.

[0049] Using the genomic DNA of the wheat to be tested as a template, and primer pairs α / β3-F and α / β3-R as amplification primers, if a DNA fragment of 180 bp can be amplified, then the wheat to be tested contains the α / β3 prolysin gene; if a DNA fragment of 180 bp cannot be amplified, then the wheat to be tested does not contain the α / β3 prolysin gene.

[0050] Using the genomic DNA of the wheat to be tested as a template, and primer pairs α / β4-F and α / β4-R as amplification primers, if a DNA fragment of 414 bp can be amplified, then the wheat to be tested contains the α / β4 prolysin gene; if a DNA fragment of 414 bp cannot be amplified, then the wheat to be tested does not contain the α / β4 prolysin gene.

[0051] Using the genomic DNA of the wheat to be tested as a template, and primer pairs α / β5-F and α / β5-R as amplification primers, if a DNA fragment of 187 bp can be amplified, then the wheat to be tested contains the α / β5 alcohol-soluble protein gene; if a DNA fragment of 187 bp cannot be amplified, then the wheat to be tested does not contain the α / β5 alcohol-soluble protein gene.

[0052] This application also provides a method for preparing processed quality improved wheat, the method comprising: reducing the expression level of the gliadin gene in wheat named wheat A to obtain processed quality improved wheat, wherein wheat A is wheat containing α / β3 gene, α / β4 gene and α / β5 gene;

[0053] The prolysin genes include α / β3, α / β4 and α / β5 genes, wherein the α / β3 gene is a DNA sequence with the nucleotide sequence of SEQ ID No. 1, the α / β4 gene is a DNA sequence with the nucleotide sequence of SEQ ID No. 2 and the α / β5 gene is a DNA sequence with the nucleotide sequence of SEQ ID No. 3.

[0054] Compared to wheat A, the processed quality-improved wheat has any of the following characteristics:

[0055] A1) Increased glutenin content in grains;

[0056] A2) Increased content of high molecular weight glutenin in grains;

[0057] A3) Increased content of low molecular weight glutenin in grains;

[0058] A4) The sedimentation value of flour prepared from grains is improved;

[0059] A5) Increased wet gluten content in flour prepared from grains;

[0060] A6) The gluten index of flour prepared from seeds is improved;

[0061] A7) Bread made from flour prepared from seeds has a higher volume;

[0062] A8) Flour prepared from seeds improves the quality of bread made from it;

[0063] A9) The content of α / β prolactin in the grains is reduced;

[0064] A10) The total alcohol-soluble protein content in the grains is reduced.

[0065] In one embodiment of this application, wheat A may be wheat cultivar 81.

[0066] The processing quality is selected from at least one of the following: glutenin content in grains, high molecular weight glutenin content in grains, low molecular weight glutenin content in grains, sedimentation value of flour prepared from grains, wet gluten content of flour prepared from grains, wet gluten index of flour prepared from grains, bread volume and bread quality of bread made from flour prepared from grains, α / β prolactin content in grains, and total prolactin content in grains.

[0067] Furthermore, in the method, the reduction in the expression level of the prolysin gene is achieved through hybridization;

[0068] The hybridization process includes hybridizing wheat M1 as the female parent and wheat A as the male parent to obtain F1 generation grains; selecting grains from the F1 generation grains that do not contain the gliadin gene and planting them, then continuously backcrossing them with wheat A as the recurrent parent to obtain backcross offspring grains; selecting grains from the backcross offspring grains that do not contain the gliadin gene and planting them, then continuously self-pollinating them to obtain wheat with improved processing quality.

[0069] In one embodiment of this application, the processed quality-improving wheat may be wheat GA2pN.

[0070] The wheat GA2pN has the accession number CGMCC No. 45699 at the China General Microbiological Culture Collection Center (CGMCC).

[0071] The wheat GA2pN material lacks three α / β prolysin genes compared to the wild-type Xiaoyan 81, namely α / β3 (SEQ ID No.1), α / β4 (SEQ ID No.2), and α / β5 (SEQ ID No.3).

[0072] The agronomic traits of GA2pN are similar to those of the wild-type Xiaoyan 81. It is a winter-growing, medium-maturing variety. Seedlings are prostrate with dark green, relatively long leaves. Plant height is 80 cm, with a compact plant type and moderate lodging resistance. The spike is spindle-shaped, awnless, with white glumes and white grains; the grains are vitreous and have good grain filling. It has strong tillering ability and a high ear-forming rate, averaging 420,000 spikes per mu (approximately 667 square meters), 34 grains per spike, and a thousand-grain weight of 40 grams. It exhibits strong winter cold resistance and resistance to late spring frosts. Flag leaf tip drying is relatively severe. It is susceptible to slow stripe rust, moderately susceptible to sheath blight and stem rust, and highly susceptible to leaf rust, powdery mildew, and Fusarium head blight.

[0073] In other words, the wheat GA2pN material has similar agronomic traits to the wild-type Xiaoyan 81. Compared with the wild-type Xiaoyan 81, the wheat GA2pN lacks three α / β gliadin genes, namely α / β3 (SEQ ID No.1), α / β4 (SEQ ID No.2), and α / β5 (SEQ ID No.3).

[0074] The wheat GA2pN (Triticum sp.) is registered with the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 45699.

[0075] Furthermore, in the method, the number of consecutive backcrosses is at least 3.

[0076] Furthermore, in the method, the number of consecutive self-crosses is at least four.

[0077] Furthermore, in the method, the number of consecutive backcrosses is 3, and the number of consecutive selfcrosses is 4.

[0078] Furthermore, in the method, the continuous backcrossing includes the step of screening for seeds that do not contain the prolysin gene in the BC1F1, BC2F1 and BC3F1 generations, respectively.

[0079] Furthermore, in the method, the continuous self-pollination includes the step of screening for seeds that do not contain the prolysin gene in the BC3F2, BC3F3, and BC3F4 generations, respectively.

[0080] The wheat M1 does not contain the gliadin gene.

[0081] The processed quality-improved wheat does not contain the alcohol-soluble protein gene.

[0082] Ion beam mutagenesis, a physical radiation technique, involves injecting an ion beam into wheat seeds as an inducing factor. Besides inducing small-segment mutations and substitutions, it can also cause large-segment DNA damage, leading to insertions, deletions, and translocations, resulting in a variety of alterations. This is one of the mutagenesis breeding methods, with deletions being the most common. The inventors used ion beam mutagenesis to screen for mutations at prolysin sites, quickly obtaining ideal deletion mutants.

[0083] This application uses ion beam radiation mutagenesis to partially delete the Gli-A2 site of prolactin, reducing the content of α / β prolactin, and can select superior allelic variants to improve the processing quality of wheat.

[0084] The terms “reduced,” “decreased,” or “reduced” are used interchangeably. For example, compared with control plants, the expression of the gliadin gene and / or the content of gliadin are reduced by at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40%, or 50%, or more. The terms “increased,” “enhanced,” or “strengthened” are used interchangeably. For example, compared with control plants, the phytosterol ratio is increased by at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40%, or 50%, or more.

[0085] In some embodiments, the control plant may be a plant that has not undergone the aforementioned manipulation, such as a plant in which the gene has not been downgraded or deleted. Methods for determining specific gene expression are known in the art, including, for example, Northern and / or Western spectroscopy.

[0086] In some embodiments, this application also relates to the application of the genes and / or proteins described herein, for example, for producing genetically modified plants to obtain improved product quality, such as flour protein properties, farinograph parameters, and extensometer parameters. In some embodiments, improved quality products can be obtained by reducing or deleting the genes in plants such as wheat.

[0087] This application describes a method for mutagenesis of mature Xiaoyan 81 seeds by ion beam radiation, followed by A-PAGE analysis of the composition of prolactin in the mutagenized offspring. The method screened for mutants with Gli-A2p sites that lack three α / β prolactin genes.

[0088] This application involved backcrossing the mutant with the wild type for three generations, followed by four generations of self-crossing, resulting in genetic material that was identical to Xiaoyan 81 in genetic background but lacked three α / β gliadin genes, which was named wheat GA2Np (Triticum sp.).

[0089] This application utilizes RNA-Seq technology to compare the transcriptome sequences of chromosome 6A of wild-type and mutant strains, and found that GA2pN lacks three α / β prolysin genes.

[0090] Based on the sequences of three α / β prolactin genes, this application developed three pairs of AS-PCR-specific primers that can identify α / β prolactin genes at the Gli-A2p site. In some embodiments, this application found that in wheat materials, using the primers selected in this application, three correctly amplified GA2pN materials lacking the three prolactin genes, namely α / β3, α / β4, and α / β5, can be obtained, and the wheat materials exhibit significant improvements in multiple quality parameters, such as sedimentation index, gluten index, and wet gluten content, which are significantly superior to those of wild-type wheat.

[0091] In some embodiments, various aspects of this application particularly relate to α / β prolysin genes and / or their expression products (i.e., the proteins they express) with deleted or downexpressed Gli-A2p sites, preferably α / β prolysin genes selected from one, two, or three of the following three α / β prolysin genes: α / β3, α / β4, and α / β5. In some embodiments, the prolysin genes described in this application include α / β3, α / β4, and α / β5. In some embodiments, various aspects of this application particularly relate to the above three genes and any combination of one, two, or three selected from the remaining three genes (and / or the proteins they express).

[0092] Compared with the prior art, this application has the following beneficial technical effects:

[0093] This application, through the determination of the protein characteristics of mutant and wild-type flour, found that the mutant GA2pN showed significant improvements in multiple quality parameters, such as sedimentation value, wet gluten content, and gluten index, which were significantly superior to those of the wild type. This application achieves the goal of significantly improving the quality of wheat dough and the baking quality of bread, and has important application value.

[0094] The mutant wheat GA2pN (Triticum sp.) obtained in this application has important application value in breeding and can be used as a high-quality breeding material.

[0095] Preservation Instructions

[0096] Material name: Wheat GA2pN

[0097] Latin name: Triticum sp.

[0098] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0099] Collection institution abbreviation: CGMCC

[0100] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0101] Deposit date: January 17, 2024

[0102] Registered with the China National Collection Center (CGMCC) No. 45699. Attached Figure Description

[0103] Figure 1 The selection process for GA2pN materials. Figure 1 In this example, B is M1 in step (3) of Example 1, and A is the wild type of Xiaoyan 81.

[0104] Figure 2 Field photographs of GA2pN and its wild-type Xiaoyan 81.

[0105] Figure 3 These are potted individual plants of GA2pN and its wild-type Xiaoyan 81.

[0106] Figure 4 A-PAGE of GA2pN and its wild-type Xiaoyan 81 prolysin.

[0107] Figure 5 MALDI-TOF-MS mass spectra of GA2pN and its wild-type Xiaoyan 8 prolysin.

[0108] Figure 6These are the RP-HPLC peaks of the alcohol-soluble protein GA2pN and its wild-type Xiaoyan 81. GA2pN, compared to wild-type Xiaoyan 81, lacks the peaks indicated by arrows 2 and 3.

[0109] Figure 7 The relative expression levels of three α / β prolactin genes in GA2pN and wild-type Xiaoyan 81 were obtained from RNA-Seq analysis.

[0110] Figure 8 PCR electrophoresis diagram of the prolysin gene primers for Chinese Spring (CS) and Chinese Spring tetrasomal deficient materials.

[0111] Figure 9 PCR electrophoresis images of the prolactin gene primers for Chinese Spring (CS), Chinese Spring tetrasomal material CSN6AT6B, Xiaoyan 81, and GA2pN.

[0112] Figure 10 Bread was prepared by baking flour from GA2pN and its wild-type Xiaoyan 81. Detailed Implementation

[0113] 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.

[0114] 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.

[0115] The NY / T 1094.1-2006 mentioned in the following examples is a currently valid agricultural standard that describes the experimental milling of wheat, Part 1: Equipment, Sample Preparation and Wheat Conditioning Operation Procedures.

[0116] The NY / T 1095-2006 mentioned in the following examples is a currently valid agricultural standard that describes the operating procedure for determining the sedimentation value of wheat using the Zeleny method.

[0117] The GB / T 5506.2-2008 standard used in the following examples is a currently valid national standard that describes the operating procedures for determining the gluten content of wheat and wheat flour, Part 2: Instrumental method for wet gluten.

[0118] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0119] Example 1: Ion beam irradiation of mature Xiaoyan 81 seeds yielded mutants lacking the Gli-A2p-null site in the α / β prolysin gene.

[0120] 1.1 Obtaining wheat GA2pN materials and their agronomic traits

[0121] Besides inducing small-segment mutations and substitutions, ion beam radiation can also cause large-segment DNA damage, leading to large-segment insertions, deletions, and translocations. Deletion is the most common, allowing for the modification of specific traits in crops, particularly for the study of gliadin sites in multi-gene families. The flowchart for preparing GA2pN materials with the Gli-A2p-null site deletion in the α / β gliadin gene is shown below. Figure 1 As shown, the specific steps are as follows:

[0122] (1) Using homozygous wheat cultivar Xiaoyan 81 self-pollinated single-seed (i.e., single-seed) seeds as material, the seeds were treated with N + Irradiation was performed with an ion energy of 30 keV and an injection dose of 8 × 10⁻⁶. 17 ions / cm 2 2020 fruit-bearing M1 plants were obtained. M1 plants were self-pollinated to produce M2 seeds. Five seeds were randomly selected from each M1 plant, cut in half, and the composition of the prolactin was determined by mass spectrometry combined with A-PAGE. The mutant at the Gli-A2p-null site was found, and half seeds homozygous for the Gli-A2p-null site were obtained.

[0123] (2) Germinate and harvest the half-seeds of the homozygous mutant Gli-A2p-null site identified in step 1 (seeds obtained by self-pollination of M2) to obtain the M3 generation, and use the A-PAGE method to identify whether they are homozygous. Germinate and harvest the half-seeds of the M3 generation that are identified as homozygous by the A-PAGE method to obtain the homozygous M4 generation seeds.

[0124] (3) The mature wheat plants obtained by germinating wheat M1 grains were used as the female parent and backcrossed three times with wild-type Xiaoyan 81 as the male parent, followed by one self-cross to obtain BC3F2 grains. The wheat grains obtained from each generation were halved and the prolactin was extracted. The grains with the deletion of the Gli-A2p-null site of the α / β prolactin gene were identified by A-PAGE.

[0125] (4) The BC3F2 seeds with the Gli-A2p-null site deletion obtained in step (3) were germinated and self-crossed for two generations to obtain BC3F3 seeds and BC3F4 seeds respectively. The purity of each generation of seeds was tested by A-PAGE. The BC3F4 seeds were planted in the field to select target single plants with agronomic traits consistent with those of the wild type of Xiaoyan 81.

[0126] The field selection method was as follows: BC3F4 grains and Xiaoyan 81 wild-type grains were planted and selected in experimental fields in Yuanyang and Maozhuang. Specifically, BC3F4 grains were sown individually in rows 2 meters long with a plant spacing of 10 centimeters, with 20 plants per row; and in rows with a row spacing of 25 centimeters, four rows were planted respectively. This was repeated three times. Management during the vegetative growth period, pollination period, and grain-filling period was the same as for field wheat. The planting of Xiaoyan 81 wild-type was the same as that of BC3F4.

[0127] (5) The target single plant obtained in step (4) is propagated (self-pollinated) to obtain wheat GA2pN material.

[0128] The wheat GA2pN material was deposited at the China General Microbiological Culture Collection Center on January 17, 2024, with accession number CGMCC No. 45699.

[0129] Phenotypic analysis was conducted on wheat GA2pN material and wild-type Xiaoyan 81 in both field cultivation and pot experiments. Field cultivation followed the planting method for BC3F4 grains. In the pot experiment, a total of 16 pots were planted, with one plant in each pot.

[0130] Field photos of GA2pN and its wild-type Xiaoyan 81, as shown. Figure 2 Photos of potted single plants of GA2pN and its wild-type Xiaoyan 81 are shown below. Figure 3 The agronomic traits of GA2pN are similar to those of the wild-type Xiaoyan 81. It is a winter-growing, medium-maturing variety. Seedlings are prostrate with dark green, relatively long leaves. Plant height is 80 cm, with a compact plant type and moderate lodging resistance. The spike is spindle-shaped, awnless, with white glumes and white grains; the grains are corneous and have good grain filling. It has strong tillering ability and a high ear-forming rate, averaging 420,000 spikes per mu (approximately 667 square meters), 34 grains per spike, and a thousand-grain weight of 40 grams. It exhibits strong winter cold resistance and resistance to late spring frosts. Flag leaf tip drying is relatively severe. It is susceptible to slow stripe rust, moderately susceptible to sheath blight and stem rust, and highly susceptible to leaf rust, powdery mildew, and Fusarium head blight.

[0131] Conclusion: The wheat GA2pN material has similar agronomic traits to the wild-type Xiaoyan 81.

[0132] 1.2 After cutting the seeds in half, extract the prolamins.

[0133] The steps for extracting prolysin after halving the seed are as follows:

[0134] (1) Select one plump seed and cut it in half. Place the half seed with the embryo in a 1.5 mL centrifuge tube for storage and use later. Place the remaining half seed with the endosperm in a 2.0 mL centrifuge tube and grind it into powder.

[0135] (2) Add 300 μL of 70% ethanol to the centrifuge tube and shake on a shaker for 1 hour.

[0136] (3) Centrifuge at 12000 rpm for 10 min and collect the supernatant. Transfer 70 μL of the supernatant to a new 1.5 mL centrifuge tube, place it in a 37 °C oven, and dry overnight to obtain alcohol-soluble protein powder.

[0137] (4) Add 60 μL of 5% acetic acid solution to the alcohol-soluble protein powder and shake gently to promote the dissolution of alcohol-soluble protein.

[0138] (5) Add 60 μL of 5% methyl acetate green loading buffer (a 5% aqueous solution of methyl acetate green containing 4.5 M urea) at a volume ratio of 1:1, and mix well. Incubate in a 70°C water bath for 5 min, and cool to room temperature to obtain the alcohol-soluble protein.

[0139] 1.3A-PAGE method for determining the composition of alcohol-soluble proteins

[0140] The procedure for determining the composition of alcohol-soluble proteins using the A-PAGE method is as follows:

[0141] (1) Preparation of A-PAGE gel: 20 mL of A-PAGE (2 10-well comb plates), 9 mL of H2O, 0.25 mL of 1.0 M Tris-HCl pH 8.5, 1 mL of acetic acid, 6 mL of 40% Acr-Bis (29:1), 6 g of urea, 400 μL of 10% APS, and 200 μL of TEMED.

[0142] (2) After mixing, put it into an ultrasonic machine and sonicate for 10 minutes.

[0143] (3) Pour into the plate, insert the comb, let stand for 60 minutes, and remove the comb after it solidifies.

[0144] (4) Pre-electrophoresis. Use 5% acetic acid as buffer (one buffer can be used up to four times), and perform electrophoresis at a reverse voltage of 150V for 1.5h.

[0145] (5) Gel running. First, use a pipette to agitate the comb wells to remove the urea inside. Then, spot the extracted alcohol-soluble protein sample (8 μL per well). Apply reverse voltages of 150V for 2 hours, 250V for 2 hours, and 400V for 4 hours.

[0146] (6) Staining and decolorization after removing the adhesive. Stain with trichloroacetic acid dye solution (0.125 g R250, 60 g trichloroacetic acid, 90 mL methanol, and dilute to 500 mL with water. The dye can be used a maximum of 3 times), and stain overnight. On the second day, decolorize with water until the base color is clean.

[0147] (7) Take a photo using a scanner.

[0148] (8) A-PAGE identification results of GA2pN and its wild-type Xiaoyan 81 prolysin are as follows: Figure 4 As shown, Figure 4 Compared to wild-type Xiaoyan 81, the GA2pN variant lacks the four bands indicated by the four arrows. Figure 4 The grains lacking the four bands indicated by the arrows are homozygous mutant grains with a deletion at the Gli-A2p-null site of the α / β prolysin gene.

[0149] 1.4 Mass spectrometry determination of alcohol-soluble protein composition

[0150] (1) Take 200 μL of the supernatant from step (3) in 1.2 and transfer it to a 1.5 mL centrifuge tube.

[0151] (2) Centrifuge the supernatant at 11,000 rpm for 5 min.

[0152] (3) Take 5 μL of supernatant, add 20 μL of loading buffer (50% (v / v) acetonitrile solution + 0.1% (v / v) trifluoroacetic acid solution mixed evenly, freshly prepared and stored at 4℃ for one day) to dilute and mix well to obtain protein dilution.

[0153] (4) Sandwich loading: 0.7 μL SA matrix (sinapic acid (SA) dissolved in the loading buffer of step 5) + 0.7 μL protein diluent + 0.7 μL SA matrix (sinapic acid (SA) dissolved in the loading buffer of step 5).

[0154] (5) Read the mass spectrum.

[0155] The MALDI-TOF-MS mass spectra of GA2pN and its wild-type Xiaoyan 8 prolactin are as follows: Figure 5 As shown, Figure 5 The results showed that wheat GA2pN lacked peak number 2 compared to wild-type Xiaoyan 81. This indicates that wheat GA2pN lacks the mass spectrometry peaks of proteins expressed by the α / β3, α / β4, and α / β5 genes.

[0156] Example 2: RP-HPLC identification of GA2pN and its wild-type Xiaoyan 81 alcohol-soluble protein

[0157] GA2pN material and wild-type Xiaoyan 81 were propagated and mature seeds were harvested. Flour was experimentally milled using a BUHLER experimental mill (DLFU; Nr: 20 355 539) according to NY / T1094.1-2006 (Experimental Wheat Milling Part 1: Equipment, Sample Preparation and Conditioning) (flour moisture content 14.0%). Equal masses of flour were weighed to extract prolamins, which were analyzed using an Agilent 1260 RP-HPLC (reversed-phase high-performance liquid chromatography). Three biological replicates were performed to analyze the peak shapes of α / β prolamins and to identify peaks missing in GA2pN.

[0158] The steps for extracting prolysin are as follows:

[0159] Weigh 40 mg of flour and place it in a 2.0 mL centrifuge tube. Add 1 mL of 70% ethanol solution and extract on a shaker for 1 hour. Centrifuge at 11,000 rpm for 10 minutes and collect the supernatant to obtain the alcohol-soluble protein solution.

[0160] RP-HPLC operating procedures:

[0161] An Agilent 1260 high-performance liquid chromatograph (HPLC) system was used, employing a Waters eAlliance HPLC system (e2695 + 2998 PDA detector). Empower 2 software was used on the workstation. A ZORBAX Eclipse Plus C18 elution column (4.6 mm × 250 mm) was used. Elution methods were as follows: eluent A (0.06% trichloroacetic acid (TFA, v / v) + H₂O), eluent B (0.05% trichloroacetic acid (TFA, v / v) + acetonitrile (ACN)); elution gradient: 0 min 21% solution B, 60 min 46% solution B, linearly increasing; flow rate 1 mL / min; column temperature 60 °C; column pressure below 160 bar.

[0162] See results Figure 6 . Figure 6 These are the RP-HPLC peaks of the alcohol-soluble protein GA2pN and its wild-type Xiaoyan 81. Figure 6 It can be seen that GA2pN lacks the chromatographic peaks indicated by arrows 2 and 3 compared to wild-type Xiaoyan 81. This indicates that wheat GA2pN lacks the chromatographic peaks of proteins expressed by the α / β3, α / β4, and α / β5 genes.

[0163] Example 3: RNA-Seq analysis of GA2pN and its wild-type Xiaoyan 81

[0164] Field sampling was conducted at Maozhuang Farm between 9:00 AM and 10:00 AM. Two replicate plots were used, selecting grains from the main stem spike of GA2pN material and wild-type Xiaoyan 81 at 10, 15, and 25 days post-flowering grain-filling stages. The grains were rapidly cooled and preserved with liquid nitrogen. Grains from each plot were randomly mixed and RNA was extracted using the RNAprep pure Plant Kit (Cat.#DP432) from Tiangen Biotech Co., Ltd. RNA concentration and integrity were assessed using Nanodrop (Thermo Fisher Scientific, Wilmington, DE) and 1% agarose gel electrophoresis. Transcriptome sequencing was performed by Beijing Berry Genomics Co., Ltd. RNA concentration and integrity were measured using an Agilent 2100 (Agilent Technologies, USA) before sequencing. Libraries were prepared after the total RNA samples passed the tests. After library construction, preliminary quantification was performed using Qubit 2.0, followed by insertion size detection using Agilent 2100. Once the insertion size met expectations, the effective concentration (>2 nM) of the library was accurately quantified using qPCR to ensure library quality. After passing the library inspection, paired-end sequencing (PE125) was performed. Transcriptome data were obtained from seeds of GA2pN material and wild-type Xiaoyan 81 at 10, 15, and 25 days post-flowering grain filling stages, with three biological replicates and a total of 18 samples. Data analysis used RSEM (1.2.25) software for read mapping, referencing CS CDS and the CDS sequence of gluten protein in Xiaoyan 81 seeds. EdgeR (3.1.3) was used to analyze differentially expressed genes in seeds of GA2pN material and wild-type Xiaoyan 81 at 10, 15, and 25 days post-flowering grain filling stages.

[0165] The wheat GA2pN material lacks three α / β prolysin genes compared to the wild-type Xiaoyan 81, namely α / β3 (SEQ ID No.1), α / β4 (SEQ ID No.2), and α / β5 (SEQ ID No.3).

[0166] The results of RNA-Seq analysis on the relative expression levels of GA2pN and three α / β prolactin genes in wild-type Xiaoyan 81 are as follows: Figure 7As shown, compared with wild-type Xiaoyan 81, the expression levels of three α / β genes, TraesCS6A01G049200, TraesCS6A01G049400, and TraesCS6A01G049500, were significantly reduced in GA2pN material at 10, 15, and 25 days after flowering. This indicates that GA2pN material lacks α / β3 (SEQ ID No. 1), α / β4 (SEQ ID No. 2), and α / β5 (SEQ ID No. 3) compared with Xiaoyan 81.

[0167] Example 4: Development of AS-PCR-specific primers

[0168] Based on the differentially expressed genes identified in Example 3, the expression levels of three prolysin genes were found to be lower in the GA2pN material compared to Xiaoyan 81: α / β3 (SEQ ID No. 1), α / β4 (SEQ ID No. 2), and α / β5 (SEQ ID No. 3). Based on the missing sequences of the three α / β prolysin genes, allele-specific PCR (AS-PCR) was designed. This is a PCR targeting a single SNP, where an artificial mismatched base is introduced to improve the efficiency of amplifying the specific fragment. The effective amplification rate of the AS-PCR primers was verified in *CS*, *CS* tetrasomal-deficient material, Xiaoyan 81, and GA2pN materials. Three primer pairs were obtained that could correctly amplify the three prolysin genes missing in the GA2pN material, amplifying α / β3, α / β4, and α / β5, respectively. The primer sequences are shown in Table 1.

[0169] Genomic DNA from *Chinese Spring* (CS), *Chinese Spring* tetrasomal-deficient material, *Xiaoyan 81*, and *GA2pN* materials was amplified by PCR using the primer pairs listed in Table 1. The PCR products were then subjected to agarose gel electrophoresis. Results are as follows: Figure 8 and Figure 9 As shown, the results indicate that the GA2pN material lacks the amplification fragments of three α / β prolysin genes.

[0170] Table 1. Primers for GA2pN material lacking the prolysin gene

[0171]

[0172] Example 5: Determination of the quality characteristics of GA2pN and its wild-type Xiaoyan 81 flour

[0173] After harvesting and drying, the seeds of GA2pN and its wild-type Xiaoyan 81 homozygous variety were milled experimentally using a BUHLER experimental mill (DLFU; Nr: 20 355 539) according to the method in NY / T1094.1-2006 (Experimental Wheat Milling Part 1: Equipment, Sample Preparation and Wheat Conditioning) (flour moisture content 14.0%). After milling, the following quality tests were performed.

[0174] 5.1 Analysis of Flour Protein Content

[0175] The quantitative analysis of flour protein uses a BSA standard curve, and the specific steps are as follows:

[0176] Preparation of phosphate-buffered saline (PBS):

[0177] (1) Solution I: 250 mL of 0.05 mol / L Na2HPO4 and 0.5% SDS (w / v) solution;

[0178] (2) Solution II: Prepare 250 mL of 0.05 mol / L NaH2PO4·H2O and 0.5% SDS (w / v); Place Solution I in a beaker and slowly add Solution II until the pH drops to 6.9 (15-20 mL will remain in Solution II). The resulting solution is PBS buffer.

[0179] (3) Weigh 1 mg bovine serum albumin (BSA) and 10 mL PBS buffer to prepare a BSA solution with a concentration of 1 mg / mL.

[0180] (4) A standard curve was prepared and plotted using a 1 mg / mL BSA standard solution. The gradient is shown in Table 2 below:

[0181] (5) The peak area of ​​the standard solution was determined by reversed-phase high-performance liquid chromatography (RP-HPLC);

[0182] (6) Use Excel to plot the standard curve and calculate the protein content of the sample.

[0183] Table 2. Gradient of BSA standard solutions

[0184] Standard Protein (BSA) 1 1 / 2 1 / 4 1 / 8 1 / 16 0 1 mg / ml BSA solution 400μL 200L 100μL 50μL 25μL 0μL PBS buffer 0μg 200μL 300μL 350μL 375μL 400μL

[0185] The results are shown in Table 3. It can be clearly seen from Table 3 that, compared with its wild-type Xiaoyan 81, GA2pN has significantly lower total prolactin content and α / β prolactin content, while its total glutenin content, high molecular weight glutenin content, and low molecular weight glutenin content have significantly increased.

[0186] Table 3. Analysis of gluten protein content in Xiaoyan 81 and GA2pN materials

[0187]

[0188] 5.2 The biuret method was used to determine the protein content.

[0189] (1). Experimental Principle

[0190] The biuret method is characterized by its simplicity, speed, and high specificity. In an alkaline copper tartrate biuret reagent, an equal volume of isopropanol is added to generate a stable solution. Protein peptide bonds form a purple-red complex with Cu²⁺ in the solution. The intensity of the color is directly proportional to the protein content, regardless of the protein's relative molecular mass or amino acid composition. For starch-rich grain samples, protein extraction is not required; the determination can be performed directly. Therefore, this method is of great significance for the rapid determination of protein content.

[0191] (2). Medicines and reagents

[0192] ① Copper sulfate pentahydrate. ② Potassium sodium tartrate. ③ Sodium hydroxide. ④ Biuret reagent: Dissolve 1.50g copper sulfate and 6.0g potassium sodium tartrate in 500mL of water, add 300mL of 10% sodium hydroxide solution while stirring, dilute with water to 1000mL, and store in a plastic bottle (or a bottle with paraffin coating on the inner wall). ⑤ 10mg / mL bovine serum albumin solution: The protein nitrogen content of bovine serum albumin should first be determined by the micro Kjeldahl method. Prepare a standard solution (using 0.05mol / L sodium hydroxide solution) according to its purity.

[0193] (3) Instruments and tools

[0194] ① 722 or other model spectrophotometer. ② Centrifuge. ③ Electronic balance with a sensitivity of 0.001g. ④ Centrifuge tubes.

[0195] ⑤ Beaker. ⑥ Pipette. ⑦ Volumetric flask.

[0196] (4). Measurement steps

[0197] ① Establishment of the standard curve: Take 12 test tubes and divide them into two groups. Add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of standard bovine serum albumin solution to each group, respectively. Make up the volume with water to 1 mL, then add 4 mL of biuret reagent to each group. Shake well and incubate in a 60℃ water bath for 5 min. Centrifuge at 4000 r / min for 10 min. Take the centrifuged liquid and perform colorimetric analysis at 540 nm. The blank is the reagent without bovine serum albumin. Take the average values ​​of the two groups and plot the standard curve with bovine serum albumin content on the x-axis and absorbance on the y-axis as the basis for quantification.

[0198] ② Accurately weigh a certain amount of sample, add 5 mL of biuret reagent, shake thoroughly, shake in a constant temperature water bath shaker at 60℃ for 5 min, centrifuge at 4000 r / min for 10 min, take the centrifuged liquid and perform colorimetric determination at 540 nm. The blank is the reagent without bovine serum albumin.

[0199] (5). Calculation of results

[0200] Sample protein content =

[0201] In the formula: X is the protein content (μg) in the sample solution obtained from the standard curve; W is the sample weight (g); V is the sample solution volume used for colorimetry (mL); V2 is the sample solution volume (mL); 100 is the conversion factor for converting the protein content to 100g of sample; 10 6 The conversion factor is used to convert g to μg.

[0202] The results are shown in Table 6. The results indicate that the total protein content of flour prepared from wheat GA2pN harvested from Yuanyang Experimental Farm was significantly higher than that of wild-type Xiaoyan 81, while there was no significant difference in total protein content between wheat GA2pN harvested from Maozhuang Experimental Farm and wild-type Xiaoyan 81.

[0203] 5.3 Determination of gluten content and Zeleny sedimentation value

[0204] The wet gluten content of flour was determined using a Perton 1500 gluten analyzer, according to GB / T 5506.2-2008 Wheat and Wheat Flour Gluten Content Part 2: Instrumental Determination of Wet Gluten. The gluten index was calculated as (gluten remaining on the sieve / total gluten) × 100%. Zeleny sedimentation value was determined according to NY / T1095-2006.

[0205] The results are shown in Table 6. The results indicate that, compared with the flour prepared from wild-type Xiaoyan 81, the wet gluten content, gluten index, and Zeleny sedimentation value of the flour prepared from wheat GA2pN were significantly increased.

[0206] 5.4 Bread Preparation Performance Scoring

[0207] 5.4.1 Bread Making Method

[0208] (1) Formula

[0209] 100g wheat flour (14% wet basis), 1.60g instant dry yeast, 1.5g salt, 6.0g sugar, 4.0g skim milk powder, 3.0g shortening, 60mL water, appropriate amount of dry malt powder, 5mL 20μg / g potassium bromate.

[0210] (2) Preparatory work

[0211] ① Connect the preheating fermentation chamber to the power supply, and turn on the main power switch, temperature control switch, and humidity control switch in sequence. The indicator light will illuminate, and the ventilation system will begin operating. Observe the temperature and humidity displayed on the temperature and humidity display. Adjust the temperature knob to the preset temperature of 30℃, and the heating element will begin heating. Turn on the humidifier switch on the left side of the chamber, and the humidifier will start operating. When the temperature reaches 30℃ and the relative humidity reaches 85%, turn off the humidifier switch (turn it on again if needed), and the system will automatically adjust to a constant temperature state.

[0212] ② Melt the shortening by heating until melted and place it in a water bath at 55-60℃.

[0213] ③ Connect the preheating oven to the power supply, turn on the oven switch and temperature control switch in sequence, the oven will start heating, adjust the temperature knob to the preset temperature of 210℃.

[0214] ④ Weighing the sample

[0215] According to the ingredient ratio of the bread baking experiment, accurately weigh the flour, yeast, malt powder and skim milk powder, put them in the fermentation bowl and mix well. Then weigh the shortening and put it on the surface of the mixed dry materials. Cover the fermentation bowl and set it aside.

[0216] ⑤ Kneading noodles

[0217] Use a pipette to draw up the salt-sugar solution and potassium bromate solution, and place them in a mixing bowl. Add the remaining water using a graduated cylinder. Add dry materials such as flour.

[0218] ⑥ Turn on the dough mixer. First, knead at low speed for about 30-60 seconds, then at high speed for about 1 minute. Stop the dough mixer, add the shortening, and knead at low speed for about 30 seconds, then switch to high speed until the dough rotates with the hook of the mixer and makes a slapping sound against the mixer wall. The gluten in the dough has reached a fully developed state. At this point, the surface of the dough is dry and shiny, delicate and clean, and feels soft and not rough. When you pull the dough by hand, it has good extensibility and elasticity. The dough is very soft and can generally be stretched into a uniform thin film.

[0219] ⑦ Carefully insert the thermometer into the middle of the dough and squeeze it tightly with your hand. After 60 seconds, read the dough temperature. The dough temperature should be (30±1)℃. The experimenter can adjust the dough temperature by controlling the water temperature and the room temperature.

[0220] (3) Fermentation and tableting

[0221] ① Remove the prepared dough from the mixing bowl, shape it into a round ball with the smooth side facing up, and place it in a lightly oiled fermentation bowl. Place it in a fermentation box and ferment for 90 minutes. The temperature of the fermentation box is (30±1)℃, and the relative humidity is 85%. The fermentation time is calculated from the moment the dough is started being mixed.

[0222] ② When the dough has fermented for 55 minutes and 80 minutes, roll it out twice. Remove the dough from the fermentation box, gently knead it until smooth and stretch it. Turn on the rolling mill, adjust the rolling gap to 0.6 cm, and roll the dough into a long sheet. Fold the sheet into 3 layers or fold it twice, place it seam-side down in the fermentation bowl, and return it to the fermentation box. Remove the dough after 90 minutes.

[0223] ③ Dividing: When the dough has fermented for 90 minutes, remove the dough from the fermentation bowl and divide it into 2 equal parts (for 200g of flour) or 3 equal parts (for 300g of flour) with scissors, and then balance it with a balance.

[0224] (4) Tableting

[0225] ① Knead the dough until smooth and stretch it appropriately. Use a sheet press to press the dough twice to make long sheets. The first roll gap is 0.7cm and the second roll gap is 0.5cm.

[0226] ②Use your hands to roll up the dough sheet from the small end. When rolling, press it down as much as possible to expel the air. Then gently roll the dough several times to make it the same size as the bread tin. Place the dough seam-side down in the oiled bread tin.

[0227] (5) Awakening

[0228] After the bread is shaped and packed into cans, it is placed in a fermentation box for proofing. The temperature of the fermentation box is (30±1)℃ and the relative humidity is 85%~90%. After proofing for 45 minutes, it is taken out.

[0229] (6) Baking

[0230] Bake the dough in the oven at a temperature of 210–230°C. Before putting the bread in the oven, place two cups of water inside to adjust the humidity. Bake for 15–25 minutes and then remove the bread from the oven (the experimenter can determine the time to remove the bread based on the color of the crust).

[0231] 5.4.2 Bread Scoring Criteria

[0232] (1) Bread volume

[0233] After the bread is baked, its volume is measured and weighed within 10 minutes, expressed in cm. Take a sample of bread to be tested, weigh it, and place it in a container of a certain volume. Add small granular filler (millet or rapeseed) to the container, completely covering the bread sample and shaking it to fill it completely. Use a ruler to level the filler, remove the bread, pour the filler into a graduated cylinder and measure its volume. Subtract the filler volume from the container volume to obtain the bread volume.

[0234] (2) Detection methods and scoring criteria for bread scoring

[0235] After the bread has cooled at room temperature for 1 hour, it is placed in a plastic bag and the bag is sealed tightly. After 18 hours, the external and internal characteristics of the bread are evaluated by sensory evaluation, which mainly includes the following: crust color, crust texture and bread shape, bread core color, bread core smoothness, bread core elasticity, bread core structure and taste.

[0236] 1) Bread volume

[0237] Bread made from different types and varieties of wheat flour varies significantly in volume, with strong gluten flour producing larger loaves than weak gluten flour. Bread volume plays a significant role in bread quality evaluation, showing a significant or highly significant positive correlation with the overall bread score and a significant positive correlation with specific volume. Therefore, bread volume is an important indicator for evaluating baking quality.

[0238] Table 4. Bread Volume Scoring Criteria

[0239] <![CDATA[Volume / cm 3 > Rating / points (out of 35) <![CDATA[Volume / cm 3 > Rating / points (out of 35) <350 0 621～650 20 351～380 2 651～680 22 381～410 4 681～710 24 411～440 6 711～740 26 441～470 8 741～770 28 471～500 10 771～800 30 501～530 12 801～830 32 531～560 14 831～860 34 561～590 16 >860 35 591～620 18

[0240] 2) Bread shape

[0241] Different types of bread have different shapes, but a normal loaf of bread should be whole in shape, with a symmetrical length, width and height, square shape, and slightly rounded edges that are not too sharp. The two ends and the middle should be neat and tidy, without any unevenness or drooping at the four ends. There should also be no cracks on the surface or broken parts in the middle or at the edges.

[0242] When baking, the ear-shaped bread will expand and form a neck and crown, resembling a mushroom. The neck should extend 3-4 cm beyond the ear and connect the top and sides in a thread-like manner, without breaking into a lid shape. The shape of the ear-shaped bread is related to the quality of the wheat. Bread baked with poor-quality wheat flour has a shorter neck and a smaller crown; while bread baked with strong gluten and fully fermented flour has a stronger expansion force during baking, resulting in a higher neck and a larger crown.

[0243] Crust Color: A normal bread crust should be golden yellow, orange-yellow, or orange. The top should be darker than the edges, and the color should be uniform, without spots or streaks. The crust color is related to baking temperature and the amount of residual sugar in the dough. If the crust is too light, it may be due to insufficient baking time, too low oven temperature, low sugar content in the recipe, low amylase activity in the flour, or too long a primary fermentation time. In the laboratory, crust color is evaluated based on the completeness of the Maillard reaction and the appropriate level of caramelization; therefore, a darker color is required, generally only differing by 1-2 points. Crust color typically accounts for 5-10 points in bread scoring.

[0244] Crust Texture: A good bread crust should be a soft and even thin layer, without any raised tops, roughness, or cracks. Slight wrinkles are acceptable as the bread shrinks when cooled. The crust texture is mainly related to the amount of oil and sugar used; too much oil or sugar will make the crust thick and tough. Over-fermentation will result in a white crust with many fragments, while under-fermentation will produce a dark brown, thick, and tough crust. Too low an oven temperature will make the crust dull, tough, and lackluster, while too high a temperature will result in a charred, cracked, and thickened crust.

[0245] Bread interior color: The normal interior color of bread should be pure white or milky white with a silky sheen. Developed countries require the interior of bread to be oily yellow or bright yellow. The interior color is related to the raw materials and processing. High-refinement flour with less bran results in a white color; insufficient gluten strength and weak gluten network structure result in large air holes, coarse particles, and a dark color; insufficient mixing, less gluten formation, and insufficient or excessive fermentation result in large, coarse bread particles, many holes, many shadows, and a dark, grayish-white interior color.

[0246] Smoothness: Smoothness reflects the fineness of the bread's texture, which is related to the processing technology and the quality of the wheat flour. Proper mixing, fermentation, and proofing result in a finer gluten network structure, smaller bread particles and thinner-walled air pockets, predominantly elongated in shape, elastic and soft, and does not crumble easily when sliced. It feels soft, smooth, and has relatively low friction. If the flour is used with insufficient force, or if mixing and fermentation are improper, the gluten network structure will be coarser and less elastic. The baked bread will have large air pockets and coarse particles, resulting in more fragments when sliced, thicker air pocket walls, poor elasticity, and a rough, uneven feel with high resistance. Large air pockets are mostly caused by improper shaping. Coarse, loose particles are mainly caused by insufficient dough mixing.

[0247] Texture and Structure: The texture and structure of bread reflect the processing characteristics and quality of the wheat flour. Different wheat flours vary greatly, therefore texture and structure are as important as bread volume, accounting for 1 / 5 of the total score for baked bread. The texture and structure of bread are related to the bread particles. Normal bread has a uniform internal structure, with consistent particle and pore size, no large holes, is soft and delicate, not undercooked, not broken, elastic, and has good air circulation. Besides the quality of the wheat flour, many other factors can affect the texture and structure of bread, such as uneven mixing, dough temperature that is too high or too low, improper fermentation time, excessively high fermentation temperature, excessive oil in the fermentation bowl that seeps into the dough, unevenly rolled dough, too much raw flour, low oven temperature, and yeast inactivation. Strict adherence to operating procedures is essential during processing.

[0248] Elasticity: The elasticity of bread depends on the quality and quantity of gluten in the wheat flour. Less gluten content results in weaker gluten strength, and the baked bread will have less elasticity; more gluten content results in stronger gluten strength, and the baked bread will have better elasticity. However, wheat flour with excessively high gluten content is too elastic and tough, and will not feel soft when pressed.

[0249] Texture: Normal bread is easy to chew, doesn't stick to your teeth, and has a delicate texture. Staple bread should have a slightly salty taste, while sweet bread should have a distinct sweetness. It should have no off-flavors, raw flour taste, or other strange tastes. The texture is also related to the quality of the wheat flour and the processing technology. Factors such as the fermentation time and baking time can all contribute to off-flavors in bread.

[0250] Aroma: The aroma of bread is produced by both the crust and the interior. The aroma of the crust is composed of the Maillard reaction, caramelization, and the malty aroma of the flour itself. Therefore, bread must be baked to a golden brown color and caramelize to develop its unique aroma. The aroma of the interior is composed of various esters formed by the alcohol, organic acids, and other chemical reactions produced during fermentation, combined with the malty aroma of the flour and the aromas of various raw and auxiliary ingredients. Normal bread should not have an excessively sour taste or other unpleasant odors.

[0251] Table 5. Scoring Criteria and Details for Bread

[0252]

[0253]

[0254] Table 6. Comparison of sedimentation value, wet gluten content, gluten index, total protein content, bread volume, and bread score of Xiaoyan 81 and GA2pN flours in the two experimental farms of Yuanyang and Maozhuang.

[0255]

[0256] Note: * indicates significance (P < 0.05); ** indicates extremely significant (P < 0.01). Data were processed using SPSS 19.0 statistical software. Experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used.

[0257] Table 7. Specific Evaluation Scores for Bread Quality

[0258]

[0259] Note: * indicates significance (P < 0.05); ** indicates extremely significant (P < 0.01). Data were processed using SPSS 19.0 statistical software. Experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used.

[0260] The results are shown in Tables 6 and 7. Figure 10As shown, the results indicate that compared to flour prepared from wild-type Xiaoyan 81, flour prepared from wheat GA2pN showed significantly improved bread volume, bread score, and processing quality parameters. This suggests that the deletion of α / β3, α / β4, and α / β5 in GA2pN can significantly improve the processing quality of gluten, dough, and bread. Specifically, the deletion of the α-gliadin gene significantly enhances the processing quality of the flour.

[0261] Table 8. Some sequences in this application

[0262]

[0263]

[0264] 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. A processed product, characterized by, The processed product is made from raw materials including wheat, wherein the wheat does not contain gliadin genes, and the gliadin genes are α / β3, α / β4, and α / β5 genes. The α / β3 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 1, the α / β4 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 2, and the α / β5 gene is a DNA molecule with the nucleotide sequence of SEQ ID No.

3. The wheat in question is wheat GA2pN, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 45699.

2. The processed article according to claim 1, characterized in that, The processed products are selected from flour, wheat starch and / or fermented products.

3. The use of wheat as described in claim 1 or 2 in the preparation of processed products.

4. The application of the wheat described in claim 1 or 2 in wheat breeding; The breeding indicators for wheat breeding include at least one of the following: increasing the glutenin content of grains, decreasing the α / β gliadin content of grains, decreasing the total gliadin content of grains, increasing the medium and high molecular weight glutenin content of grains, increasing the low molecular weight glutenin content of grains, increasing the sedimentation value of flour made from wheat grains, increasing the wet gluten content of flour made from wheat grains, increasing the gluten index of flour made from wheat grains, increasing the bread volume of bread made from flour made from wheat grains, and improving the quality of bread made from flour made from wheat grains.

5. A method for preparing wheat with improved processing quality, characterized in that: The method includes: deleting the gliadin gene in wheat named wheat A to obtain wheat with improved processing quality, wherein wheat A is wheat containing α / β3 gene, α / β4 gene and α / β5 gene; The prolysin genes are α / β3, α / β4, and α / β5 genes. The α / β3 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 1, the α / β4 gene is a DNA molecule with the nucleotide sequence of SEQ ID No. 2, and the α / β5 gene is a DNA molecule with the nucleotide sequence of SEQ ID No.

3. Compared to wheat A, the processed quality-improved wheat has any of the following characteristics: A1) Increased glutenin content in grains; A2) Increased content of high molecular weight glutenin in grains; A3) Increased content of low molecular weight glutenin in grains; A4) The sedimentation value of flour prepared from grains is improved; A5) Increased wet gluten content in flour prepared from grains; A6) The gluten index of flour prepared from seeds is improved; A7) Bread made from flour prepared from seeds has a higher volume; A8) Flour prepared from seeds improves the quality of bread made from it; A9) The content of α / β prolysin in the grain is reduced; A10) The total alcohol-soluble protein content in the grains is reduced; The deletion of the prolysin gene was achieved through hybridization; The hybridization process involves using wheat A as the male parent and wheat M1 as the female parent to hybridize and obtain F1 generation grains. Grains without the gliadin gene are selected from the F1 generation grains and planted, then continuously backcrossed with wheat A as the recurrent parent to obtain backcross offspring grains. Grains without the gliadin gene are selected from the backcross offspring grains and planted, then continuously self-crossed to obtain wheat with improved processing quality. The wheat M1 does not contain the gliadin gene, and the wheat M1 is wheat GA2pN.

6. The method according to claim 5, characterized in that: The number of consecutive backcrosses is at least 3.

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