Method for rapidly breeding new variety of wheat with high resistant starch based on four specific characters

Abstract

The invention relates to the technical field of breeding of new varieties of crops, in particular to a method for rapidly breeding a new variety of wheat with high resistant starch based on four specific characters. According to the breeding method provided by the invention, the high-resistance starch variety is rapidly, accurately and successfully bred on the basis of EMS mutagenesis and selection of multiple specific physical and quality characters of different generations, the Shannong RS1 # with the resistant starch content up to 11.8% and the amylose content up to 60% or above is obtained through the method, breeding only takes 6 years, and the breeding period is shortened by 4 years or above.

Description

Technical Field

[0001] The present invention relates to the technical field of breeding new crop varieties, and specifically relates to a method for rapidly breeding a new variety of high-resistant starch wheat based on four specific traits. Background Art

[0002] Like other grains, the total starch content of wheat accounts for about 70% of the grain, among which amylose and amylopectin account for 25% and 75% of the total starch respectively. After being eaten, it is quickly converted into glucose, so it is a high glycemic index (GI value about 90) food. High amylose (resistant) starch is a high-quality dietary fiber that is not decomposed into sugar in the small intestine and has obvious effects of slowly increasing blood sugar, preventing constipation and colon cancer. It has great social demand and economic benefits. However, the content of resistant starch in common wheat varieties is very low (less than 1%), so it is of great significance to cultivate a new variety of high-resistant (high amylose) starch wheat with a low blood sugar-raising effect.

[0003] For the breeding of new wheat varieties, methods such as conventional hybridization, gene editing and chemical mutagenesis are generally used. Due to the lack of high-resistant starch germplasm in nature, it is very difficult to breed high-resistant starch varieties by conventional hybridization; gene editing changes the starch synthesis gene sequence, and breeding high-resistant starch varieties is a new technology, but it requires special experimental conditions, and the linkage effects of knocking out some genes on other traits also need to be identified for many years; ethyl methanesulfonate (EMS) mutagenesis can theoretically induce base mutations and increase the amylose content, but many random mutations generated by EMS treatment require a large amount of work and time to identify and recognize. Therefore, the process of breeding high-resistant starch varieties solely by EMS mutagenesis is long and difficult.

[0004] Therefore, there is an urgent need to develop a method for breeding new varieties of high-amylose and high-resistant starch wheat. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the related technologies to some extent. For this reason, an object of the present invention is to provide a method for rapidly breeding a new variety of high-resistant starch wheat. The technical core of which is based on EMS mutagenesis, and the key lies in the selection of multiple specific physical and quality traits in different generations to rapidly and accurately breed a high-resistant starch variety. By this method, the breeding of "Shannong RS1" with a resistant starch content as high as 11.8% only took 6 years, shortening the breeding cycle by more than 4 years.

[0006] For this reason, the first aspect of the present invention provides a method for breeding a new variety of high-amylose and high-resistant starch wheat. In some embodiments of the present invention, the breeding method includes:

[0007] S1: Mutagenize the parental wheat seeds with EMS, and plant the mutagenized seeds M0 to obtain M1 seeds harvested by plant lines;

[0008] S2: Screening the strains to which the M1 seeds with wrinkled and dull seed coat belong, and planting them to obtain M2 seeds harvested according to the strains;

[0009] S3: screening the strains to which at least 90% of the M2 seeds with wrinkled and dull seed coats belong, and planting them to obtain M3 seeds harvested according to the strains;

[0010] S4: The M3 seeds are stained with I2-KI to screen out the strain to which the whole grain is stained blue-black, and at least a portion of the seeds of the strain are tested for amylose and resistant starch to determine the strain with an amylose content of at least 45% and / or a resistant starch content of at least 6%, so as to obtain a new variety of wheat with high amylose and high resistant starch.

[0011] The breeding method of the new wheat variety with high amylose and high resistant starch provided by the present invention is based on the fact that the inventor has discovered the judgment (or breeding) index for breeding high amylose and resistant starch that has not been reported or discovered and applied by the previous researchers. Specifically, the first breeding index discovered by the inventor is "shrunk and dull skin". The inventor has found that when the parent wheat seeds are subjected to mutagenesis treatment using EMS, a small amount (about 10%) of grains with shrunk and dull skin appear in the M1 seeds. This is because there are many random point mutations caused by EMS treatment, and many induced varieties do not necessarily have the phenomenon of seed coat shrinkage. In addition, most of the EMS (ethyl methanesulfonate) mutagenesis breeding conducted by the previous researchers is to obtain good agronomic and quality traits through mutation, and the seed coat shrinkage variation is not paid attention to or is eliminated as an inferior mutation. Based on the need to select special starch mutants, the present invention attaches importance to and for the first time uses the trait of shrunk and dull skin as an important index for breeding high amylose (resistant) starch.

[0012] The second breeding index discovered by the inventors is "I2-KI whole grain dyed blue", that is, it was found that with I2-KI dyeing, the whole shrunken grains turned blue-black within 1 minute, while the smooth and plump seeds were not dyed for a long time. The previous researchers used the seed section half-grain I2-KI dyeing method to select waxy wheat varieties with amylopectinase mutations, because only seeds with full mutations in the three genes of amylose synthase (GBSS), that is, full waxy wheat seeds, have purple-red cross-sections, while seeds with non-full mutations in the three genes, that is, seeds with only a small amount of amylose, have half-grain cross-sections that are blue when stained with I2-KI. The I2-KI dyeing method has never been used to select high amylose (resistant) starch varieties, so previous researchers have never reported that whole wheat is blue-black when stained with I2-KI.

[0013] In some embodiments of the present invention, the breeding method further comprises:

[0014] S5: Continuously plant the lines to which the M3 seeds with I2-KI staining being blue-black belong for at least 1 generation, detect the amylose and resistant starch contents of the seeds harvested by line, and determine the lines with amylose content reaching at least 45% and / or resistant starch content reaching at least 6% in order to obtain new wheat varieties with high amylose and high resistant starch.

[0015] In some embodiments of the present invention, the breeding method further includes:

[0016] S6: Continuously plant the lines to which the M3 seeds with whole grain I2-KI staining being blue-black belong to obtain M4 seeds harvested by line;

[0017] S7: Conduct dough water absorption rate detection on the M4 seeds harvested by line, and screen the lines to which the grains with dough water absorption rate reaching at least 75 mL belong, which are the new wheat varieties with high amylose and high resistant starch,

[0018] Or, plant the lines to which the grains with dough water absorption rate reaching at least 75 mL belong for at least 1 generation in order to obtain new wheat varieties with high amylose and high resistant starch.

[0019] The third breeding index discovered by the inventor is high water absorption rate. It is well-known in the art that the dough water absorption rate of common wheat varieties with a straight-chain to branched-chain starch ratio of 25:75 is generally about 60 mL, and the water absorption rate of waxy wheat with a branched-chain starch content as high as over 98% increases to about 70 mL. By this inference, the water absorption rate of high amylose varieties should be low, but this study found that the water absorption rate of high amylose varieties is instead as high as about 80%. Since there are few high amylose (resistant starch) wheat varieties bred at home and abroad currently, there has been no report on the phenomenon of significantly increased water absorption rate in high amylose varieties (resistant starch wheat) by predecessors, and it has not been used as an important index for selecting high amylose varieties (resistant starch wheat).

[0020] In some embodiments of the present invention, the breeding method further includes:

[0021] S8: Continuously plant the lines to which the M3 seeds with whole grain I2-KI staining being blue-black belong to obtain M4 seeds harvested by line;

[0022] S9: Conduct dough water absorption rate detection on the M4 seeds harvested by line, screen the lines to which the grains with dough water absorption rate reaching at least 75 mL belong and continue to plant them to obtain M5 seeds harvested by line;

[0023] S10: Conduct morphological identification of A starch granules on the M5 seeds harvested by line, and screen the lines to which the seeds with partially irregularly variant A starch granule shapes belong, which are the new wheat varieties with high amylose and high resistant starch,

[0024] Alternatively, the strains to which the screened A starch granules with irregular shapes belong are planted for at least one generation to obtain new wheat varieties with high amylose and high resistant starch.

[0025] Furthermore, the fourth breeding index discovered by the inventors is the irregular shape of the A starch granules of the grains. Specifically, the present invention is the first to use the irregular shape of the A starch granules observed by scanning electron microscopy, with some starch granules being elongated and twisted, in the shape of long strips or crescents, i.e. sickle-shaped, as the fourth specific trait for the base mutation of starch branching enzyme SBE and the selection of resistant starch, because the gene mutation will definitely affect the change of the morphological structure of starch granules.

[0026] In some embodiments of the present invention, the breeding method further comprises:

[0027] The breeding method further includes, in step S10, planting the strains to which the screened seeds with irregularly shaped A starch granules belong for at least one generation, detecting the contents of amylose and resistant starch in the seeds harvested from the strains, and determining the strains with an amylose content of at least 45% and / or a resistant starch content of at least 6%, so as to obtain new wheat varieties with high amylose and high resistant starch.

[0028] In some embodiments of the present invention, the concentration of EMS during the mutagenesis treatment is 0.5-1.0 mol / L.

[0029] In some embodiments of the present invention, the time of the mutagenesis treatment is 8-24 hours.

[0030] In some embodiments of the invention, the parental wheat seeds are derived from distantly related wheat varieties.

[0031] In some embodiments of the present invention, the distantly related wheat varieties include PH82-2-2 and Shannong 20.

[0032] In some embodiments of the present invention, the number of parent wheat seeds is at least 1,000, preferably 1,000-10,000.

[0033] The second aspect of the present invention provides wheat. In some embodiments of the present invention, the wheat is obtained by incrementally stacking the breeding method described in the first aspect.

[0034] In some embodiments of the present invention, the wheat is Shannong RS1, and the preservation number is CGMCC NO.:30792.

[0035] Shannong RS1 with the preservation number of CGMCC NO.: 30792 was preserved in the China General Microbiological Culture Collection Center (CGMCC, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on February 28, 2025.

[0036] The third aspect of the present invention provides the use of the wheat obtained by the breeding method described in the first aspect in the preparation of starch products.

[0037] The fourth aspect of the present invention provides a starch product. In some embodiments of the present invention, the starch product is prepared from wheat with a amylose content of at least 45% and a resistant starch content of at least 6%.

[0038] In some embodiments of the present invention, the starch product is prepared from wheat obtained by incrementally stacking the breeding method described in the first aspect.

[0039] The fifth aspect of the present invention provides a method for preparing a starch product. In some embodiments of the present invention, the preparation method includes:

[0040] A. Cultivate the wheat variety obtained by the breeding method described in the first aspect and harvest the seeds;

[0041] B. Process the seeds to obtain a starch product.

[0042] The sixth aspect of the present invention provides a method for cultivating a new wheat variety with high amylose and high resistant starch. In some embodiments of the present invention, it is obtained by at least one of the following methods:

[0043] (1) Use the wheat variety obtained by the breeding method described in the first aspect as a parent for cultivation;

[0044] (2) Gene editing;

[0045] (3) Cross breeding.

[0046] The method for breeding a new wheat variety with high amylose (resistant) starch based on the early deadline provided by the present invention has its technical core based on EMS mutagenesis, with the focus on the selection of specific physical and quality traits appearing in different selection generations, and the determination of the detection and identification of high-value starch and resistant starch. The inventor uses this technology to quickly breed new high-resistant starch varieties such as Shannong RS1 with an amylose content of about 45% and a resistant starch content of about 12%, which is at the leading level in the same field.

[0047] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the practice of the present invention.

[0048] Preservation information:

[0049] Material Name: Shanong RS1 Wheat (Triticum aestivum)

[0050] Deposit Date: February 28, 2025

[0051] Depositary Institution: China General Microbiological Culture Collection Center (CGMCC)

[0052] Deposit Number: CGMCC NO.: 30792 Description of the Drawings

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0054] Figure 1 Shows the technical route of a method for rapidly breeding new wheat varieties with high resistant starch based on four specific traits according to an embodiment of the present invention;

[0055] Figure 2 Shows the shrunken and dull-surfaced grains and normal grains in PH82-2-2M1 seeds, and the shrunken and dull-surfaced grains account for about 10%;

[0056] Figure 3 Shows the shrunken variant grains (upper row) and smooth and plump grains (lower row) in PH82-2-2M1 seeds;

[0057] Figure 4 Shows the M2 seeds obtained by planting the shrunken grains of M1. Among them, the right figure shows the grains that have inherited the shrunken characteristics by more than 90%, and the left figure shows the M2 seeds with smooth and plump seed coats obtained from the non-shrunken grains;

[0058] Figure 5A Shows the staining results of the cross-section of fully waxy wheat grains after staining with I2-KI;

[0059] Figure 5B Shows the staining results of the cross-section of normal common wheat seeds after staining with I2-KI;

[0060] Figure 6 Shows the M3 seeds obtained by planting the shrunken grains of M2. Among them, the shrunken grains are completely blue-black in the I2-KI solution, and the smooth grains are not stained;

[0061] Figure 7 Shows the determination results of the high water absorption rate of the dough of the M4 seeds obtained by planting the shrunken grains of M3;

[0062] Figure 8 Shows the determination results of the high water absorption rate of the dough of the seeds with plump and shiny seed coats obtained by planting M3;

[0063] Figure 9 Shows the starch morphology of M5 seeds determined by scanning electron microscopy, where A and B in the figure refer to A starch granules and B starch granules respectively;

[0064] Figure 10 Shows the starch morphology of PH82-2-2 control seeds determined by scanning electron microscopy, where A and B in the figure refer to A starch granules and B starch granules respectively;

[0065] Figure 11 Shows the amylose determination results of Shannong RS1;

[0066] Figure 12 Shows the resistant starch determination results of Shannong RS1. Detailed implementation manners

[0067] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0068] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality" is two or more.

[0069] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0070] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0071] Term

[0072] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0073] In this text, the terms "optionally", "optional", or "option" generally mean that the subsequent described event or condition can but does not necessarily occur, and this description includes the cases where the event or condition occurs and the cases where the event or condition does not occur.

[0074] In this text, "Amylose" is a linear polymer composed of α-D-glucose units linked by α-1,4-glycosidic bonds. Its molecular structure is relatively simple without branches. The degree of polymerization (DP) of amylose is usually between 100 and 2000, which means that an amylose molecule can consist of 100 to 2000 glucose units. In natural starches, the content of amylose usually accounts for 25% to 30% of the total starch, but there are significant differences in its content among starches from different sources. For example, the amylose content in wheat starch is about 20%-30%. During the synthesis of starch, the content and structure of amylose are affected by plant genotypes and growth environments. Amylose has a relatively high gelatinization temperature and anti-swelling property, and shows a blue reaction when reacting with iodine.

[0075] In this text, "Amylopectin" is a highly branched polysaccharide composed of glucose molecules linked by α-1,4-glycosidic bonds and α-1,6-glycosidic bonds. This branched structure makes amylopectin present a dendritic morphology, with multiple non-reducing ends and one reducing end. The molecular structure of amylopectin includes a linear part and a branched part. The linear part is linked by α-1,4-glycosidic bonds, while the branched part forms branches through α-1,6-glycosidic bonds. Each branch point appears approximately every 6-9 glucose residues, and each branch contains an average of about 15-18 glucose residues. The molecular weight of amylopectin is relatively large, generally consisting of 1000 to 300,000 glucose units.

[0076] In this text, "resistant starch" is a special form of starch, whose structure and properties make it difficult to be decomposed by digestive enzymes in the human small intestine. The formation of resistant starch is closely related to the content of amylose. Starches with a high amylose content are more likely to form resistant starch. For example, high-amylose corn starch can still maintain a relatively high level of resistance after gelatinization. In addition, by physically or chemically treating amylose, the content of resistant starch can also be increased. Resistant starch has a relatively low energy value and can be fermented by intestinal microorganisms to produce short-chain fatty acids, which helps to regulate blood sugar, lower blood lipids, and promote intestinal health.

[0077] There is a significant positive correlation between the amylose content and the resistant starch content. Starches with a high amylose content are more likely to form resistant starch, especially after treatments such as aging. In addition to the amylose content, the formation of resistant starch is also affected by factors such as the molecular structure and average degree of polymerization of starch.

[0078] "Starch products" refer to products processed from plants such as cereals, tubers, and legumes through processes including cleaning, grinding, separation, pulping, drying, and shaping. In this invention, it mainly refers to products processed from wheat obtained by the breeding method of this invention, such as low-GI meal replacement foods like biscuits and protein bars.

[0079] In this article, "distant blood relationship" refers to the genetic differences between individuals or species with relatively distant genetic relationships in genetics and breeding. In plant breeding, distant hybridization refers to using parents with relatively large genetic differences for hybridization to create new genetic combinations and excellent traits.

[0080] In this article, "wheat variety with distant blood relationship" refers to a wheat variety obtained by distant hybridization technology, which hybridizes wheat with other wild related species to introduce excellent genes and cultivate a wheat variety with new traits. For example, PH82-2-2 was developed by the Wheat Quality Breeding Group of Shandong Agricultural University using the distant hybridization material (ROF 3) from the Northwest Institute of Botany, Chinese Academy of Sciences, after treatment with 60Co-γ rays and years of selection. And ROF 3 was obtained by Chen Shuyang et al. from the former Northwest Institute of Botany, Chinese Academy of Sciences (now Northwest A&F University) through distant hybridization between wheat and Psathyrostachys huashanica (Triticum aestivum×Psathyrostachys huashanica).

[0081] In this article, "strain" refers to a group of offspring propagated from the same plant. By single-plant selection and planting, different strains can be formed for the identification and isolation of pure lines. For example, in cross-breeding, by selecting excellent single plants and planting their offspring to form strains, and then screening out excellent strains with relatively homogeneous genetics.

[0082] In this article, "increasing superposition" means that the detection effects of 4 specific traits in practical applications are increasing and superposed. For example, the I2-KI staining of M3 seeds is carried out on the basis of the detection of the first specific trait of wrinkled and dull seed coats in M1 and M2. "Wrinkled and dull seed coat + the whole grain dyed blue-black" not only increases in selective traits but also ensures the superposition of the homologous gene mutation sites of starch branching enzyme (SBE) TaSBEIIa-A, TaSBEIIa-B, and TaSBEIIa-D and the effect of controlling starch content. In EMS chemical mutagenesis, one or more base mutations may occur in these 3 homologous genes. The superposition selection of 4 specific traits in this invention is beneficial to the superposition of multiple mutation sites of these 3 homologous genes and their effects.

[0083] In this article, "Gene Editing" refers to a technology that can precisely modify the genome of an organism. By performing operations such as insertion, deletion, and replacement on the DNA sequence, the genetic information and phenotypic characteristics of the organism can be changed.

[0084] In this article, "hybrid breeding" is a method that involves artificially controlled sexual hybridization. Two or more parents with different excellent traits are mated, enabling the recombination of the genes of both parents. As a result, new genotypes and excellent traits are produced in the offspring, and new varieties are obtained through selection and cultivation. Its core principle is gene recombination. In this way, the excellent traits of multiple parents can be concentrated to form offspring with heterosis.

[0085] According to a specific embodiment of the present invention, the present invention provides a method for breeding a new variety of wheat with high amylose and high resistant starch, including:

[0086] S1: Mutagenize the parental wheat seeds with EMS and plant the mutagenized seeds M0 to obtain M1 seeds harvested by plant line.

[0087] S2: Screen the plant lines to which the M1 seeds with shrunken and dull seed coats belong, and plant them to obtain M2 seeds harvested by plant line.

[0088] S3: Screen the plant lines to which at least 90% of the M2 seeds with shrunken and dull seed coats belong, and plant them to obtain M3 seeds harvested by plant line.

[0089] S4: Stain the M3 seeds with I2-KI, screen the plant lines to which the grains that are entirely stained blue-black belong, and detect the amylose and resistant starch content of at least a part of the seeds of this plant line to determine the plant lines with an amylose content of at least 45% and / or a resistant starch content of at least 6% in order to obtain a new variety of wheat with high amylose and high resistant starch.

[0090] According to a specific embodiment of the present invention, the breeding method further includes:

[0091] S5: Continuously plant the plant lines to which the M3 seeds stained blue-black with I2-KI belong for at least 1 generation, detect the amylose and resistant starch content of the seeds harvested by plant line, and determine the plant lines with an amylose content of at least 45% and / or a resistant starch content of at least 6% in order to obtain a new variety of wheat with high amylose and high resistant starch.

[0092] According to a specific embodiment of the present invention, the breeding method further includes:

[0093] S6: Continue to plant the lines to which the M3 seeds with the whole grain stained blue - black by I2 - KI staining belong, and obtain M4 seeds harvested by line;

[0094] S7: Perform dough water absorption rate detection on the M4 seeds harvested by line, and screen the lines to which the grains with a dough water absorption rate of at least 75 mL belong, which are the new varieties of high - amylose and high - resistant - starch wheat.

[0095] Alternatively, plant the lines to which the grains with a dough water absorption rate of at least 75 mL belong for at least 1 generation to obtain the new varieties of high - amylose and high - resistant - starch wheat.

[0096] It should be noted that the breeding method provided by the present invention can directly detect the amylose and resistant starch of the seeds of the lines to which the grains stained blue - black by I2 - KI staining belong in step S4, determine the lines with an amylose content of at least 45% and / or a resistant starch content of at least 6%, so as to obtain the new varieties of high - amylose and high - resistant - starch wheat; or continue to plant the lines to which the grains stained blue - black by I2 - KI staining belong for at least 1 generation, and then perform the detection of amylose and resistant starch, determine the lines with an amylose content of at least 45% and / or a resistant starch content of at least 6%, so as to obtain the new varieties of high - amylose and high - resistant - starch wheat; or continue to plant the lines to which the grains stained blue - black by I2 - KI staining belong to obtain M4 seeds harvested by line. When a sufficient amount of seeds is obtained, perform dough water absorption rate detection on the M4 seeds harvested by line, and screen the lines to which the grains with a dough water absorption rate of at least 75 mL belong, which are the new varieties of high - amylose and high - resistant - starch wheat. Alternatively, plant the lines to which the grains with a dough water absorption rate of at least 75 mL belong for at least 1 generation to obtain the new varieties of high - amylose and high - resistant - starch wheat. All of the above three breeding routes can obtain the new varieties of high - amylose and high - resistant - starch wheat.

[0097] According to a specific embodiment of the present invention, the breeding method further includes:

[0098] S8: Continue to plant the lines to which the M3 seeds with the whole grain stained blue - black by I2 - KI staining belong, and obtain M4 seeds harvested by line;

[0099] S9: Perform dough water absorption rate detection on the M4 seeds harvested by line, and screen the lines to which the grains with a dough water absorption rate of at least 75 mL belong and continue to plant them to obtain M5 seeds harvested by line;

[0100] S10: Morphologically identify the A starch granules in the M5 seeds harvested by strain, and screen out the strains to which the seeds with irregularly variant A starch granule shapes belong, which are the new varieties of high amylose and high resistant starch wheat.

[0101] Alternatively, plant the strains to which the seeds with irregular A starch granule shapes belong for at least 1 generation to obtain new varieties of high amylose and high resistant starch wheat.

[0102] In some embodiments of the present invention, the breeding method further includes:

[0103] The breeding method further includes that in step S10, after planting the strains to which the seeds with irregular A starch granule shapes belong for at least 1 generation, detect the amylose and resistant starch contents of the seeds harvested by strain, and determine the strains with amylose content reaching at least 45% and / or resistant starch content reaching at least 6% to obtain new varieties of high amylose and high resistant starch wheat.

[0104] According to a specific embodiment of the present invention, there is no particular limitation on the concentration of EMS during the mutagenesis treatment. For example, it can be EMS with a concentration of 0.5 - 1.0 mol / L. Preferably, for the breeding method provided by the present invention, the concentration of EMS used is 0.8 mol / L. There is no particular limitation on the time of the mutagenesis treatment. For example, it can be 8 - 24 h. Preferably, for the breeding method provided by the present invention, the time for mutagenesis with EMS is 24 h.

[0105] According to a specific embodiment of the present invention, the parental wheat seeds are derived from wheat varieties with distant genetic backgrounds.

[0106] According to a specific embodiment of the present invention, the wheat variety with a distant genetic background can be PH82 - 2 - 2, or other wheat varieties with distant genetic backgrounds, which can all be used as the source of the parental wheat seeds in the breeding method of the present invention.

[0107] According to a specific embodiment of the present invention, there are at least 1000 parental wheat seeds, preferably 1000 - 10000 seeds.

[0108] According to a specific embodiment of the present invention, the present invention also provides a kind of wheat, which is obtained by incrementally stacking through the foregoing breeding method.

[0109] According to a specific embodiment of the present invention, the wheat is Shanong RS1, and the preservation number is CGMCC NO.: 30792.

[0110] The wheat obtained by the breeding method provided by the present invention can be used for preparing starch products.

[0111] According to a specific embodiment of the present invention, the present invention also provides a starch product, which is prepared from wheat with a amylose content of at least 45% and a resistant starch content of at least 6%.

[0112] According to a specific embodiment of the present invention, the starch product is prepared from wheat obtained by incrementally superimposing the breeding method described above.

[0113] According to a specific embodiment of the present invention, the present invention also provides a method for preparing a starch product, comprising:

[0114] A. Cultivate the wheat variety obtained by the breeding method described above and harvest the seeds;

[0115] B. Process the seeds to obtain a starch product.

[0116] According to a specific embodiment of the present invention, the present invention also provides a method for cultivating a new wheat variety with high amylose and high resistant starch, which is obtained by at least one of the following methods:

[0117] (1) Use the wheat variety obtained by the breeding method described above as a parent for cultivation;

[0118] (2) Gene editing;

[0119] (3) Cross breeding.

[0120] According to a specific embodiment of the present invention, the present invention provides a method for quickly breeding a new wheat variety with high resistant starch based on four specific traits, as Figure 1 shown, and the steps are as follows:

[0121] According to the breeding goal, select varieties (lines) from different sources as EMS mutagenesis materials. For the M1 seeds obtained from the planting in the mutagenesis year and the M2 seeds obtained in the next year, select them respectively according to whether the seeds show epidermal shrinkage and dull luster variation and stable inheritance (the first specific trait, Figures 2 - 4 ); for the M3 seeds obtained in the next year, select them according to the whole grain showing blue-black color by I2-KI staining (the second specific trait, Figure 6 ); for the M4 seeds obtained in the next year, select them according to the dough water absorption rate significantly increasing (the third specific trait, Figure 7 ) to more than 75 ml; for the M5 seeds obtained in the next year, select them according to the degree of partial starch grains changing from regular elliptical shape to irregular crescent shape under scanning electron microscope (the fourth specific trait, Figure 9)Make a selection, measure the resistant starch content of the selected varieties, which increases from about 1% of common wheat varieties to about 6% and is finally confirmed. It solves the technical problems of numerous random mutations generated by EMS mutagenesis, which require a large amount of work and time for identification and recognition, as well as the difficult and long scientific problem of cultivating resistant starch varieties, shortens the breeding cycle by more than 4 years, and is accurate and efficient.

[0122] According to a more specific embodiment of the present invention, the present invention provides a method for rapidly breeding new high-resistant starch wheat varieties based on four specific traits, and the steps are as follows:

[0123] Select varieties (germplasms) from different sources as materials for EMS (ethyl methanesulfonate) treatment, and select lines according to whether the M1 seeds obtained from the mutagenized materials planted in the current year and harvested in the next year show the variation of epidermal shrinkage and lack of luster ( Figure 2 、 3 ); for the M2 seeds obtained from the selected M1 seeds in the next year, classify and select them according to the first specific trait that more than 90% of the seeds stably inherit the epidermal shrinkage and lack of luster variation, Figure 4 ); for the M3 seeds obtained from the M2 seeds in the next year, select them according to the overall blue-black color of the grains stained with I2-KI (the second specific trait, Figure 6 ); for the M4 seeds obtained from the selected M3 seeds in the next year, confirm them according to the significant increase in dough water absorption rate (the third specific trait, Figure 7 ) to about 75 ml; for the M5 seeds obtained from the M4 seeds in the next year, confirm them according to the degree of change of the configuration of some starch grains from regular elliptical to irregular crescent shape under scanning electron microscopy (the fourth specific trait, Figure 9 ); for the M6 lines obtained from the confirmed M5 seeds in the next year, detect the amylose content and resistant starch content, and the two increase from about 25% and 1% of common wheat varieties to about 45% and 6% respectively, and confirm the successful cultivation of resistant starch wheat varieties.

[0124] According to a specific embodiment of the present invention, the following method can be used to perform EMS mutagenesis on seeds to rapidly breed new high-resistant starch wheat varieties based on four specific traits:

[0125] Select 6 EMS mutagenesis materials containing distant bloodlines, local germplasms and foreign germplasms, namely PH82-2-2 (distant bloodline), Shannong 20 (distant bloodline), Sankecun (local variety), GB16 (Germany), GB25 (Italy) and Mo99 (CIMMY). The specific method is as follows: select 1000 grains for each of the 6 varieties (the number of grains can be increased to improve the mutagenesis frequency). First, soak them in distilled water for 8 hours, and then place them in 0.8 mol EMS solution for 16-hour mutagenesis treatment, with at least 3 shakes during this period. When the soaking treatment is completed, repeatedly rinse the mutagenized wheat grains with clean water to obtain mutagenized wheat M0. Put the M0 seeds into a germination tray and place them in a constant temperature and humidity incubator at 20-22 °C for a 72-hour germination rate test, and rinse them with clean water every 8 hours during this period. According to the germination rate of the variety, obtain the lethality of the variety after mutagenesis treatment. Thereafter, directly sow and plant the M0 materials of the 6 varieties in the same year. Calculate the emergence rate, emergence potential and seedling growth status 15 days after emergence, so as to predict the subsequent mutagenesis results, and do a good job in the management of fertilizer, water, disease prevention and pest control during the entire growth period.

[0126] Harvest the M1 seeds obtained from the M0 planted in the previous year in June of the following year. For the M1 seeds, first, according to the possibility that starch synthase mutation may lead to changes in seed appearance, conduct an identification test of external physical traits. It is found that a small amount (about 10%) of epidermal shrinkage and dullness variations appear in the M1 seeds of PH82-2-2, and there are no obvious seed coat shrinkage variations in the other 5 seeds mutagenized at the same time. Therefore, according to whether there are epidermal shrinkage and dullness variations in the M1 seeds, they are divided into seed coat variant seeds and non-variant seeds. The M1 seeds of PH82-2-2 belong to seed coat variant seeds, and they are divided into shriveled and dull variant grains and plump and shiny non-variant grains and stored and planted separately. The M1 seeds of Sankecun, Shannong 20, GB16, GB25 and Mo99 in the same year belong to non-seed coat variant seeds. In the following year, plant the M1 seed coat variant seeds of PH82-2-2 single-grain, and plant 100 grains of non-variant seeds and the other 5 seeds as controls respectively.

[0127] Harvest the M2 seeds obtained from M1 planted in the previous year in June of the second year. Conduct seed appearance physical property identification and inspection on the M2 seeds in the same way as M1 in the previous year. It is identified that more than 90% of the M2 seeds obtained from the shriveled grains of M1 have inherited the trait of shriveled and dull seed epidermis. Thus, the trait of shriveled and dull seed epidermis is confirmed as the first specific trait index for the selection of starch branching enzyme base mutation to produce high amylose (resistant) starch. According to this trait index, the lines with high heritability of the shriveled and dull seed epidermis are selected for planting in the next year, and the M2 seeds without the trait of shriveled seed epidermis are unselected lines. The M2 seeds of PH82-2-2 with more than 90% shriveled seed epidermis trait belong to the seeds of the selected lines for planting in the next year, and the PH82-2-2 seeds with low heritability of the shriveled seed epidermis trait and the other 5 mutagenized seeds in the same period belong to the seeds of unselected varieties. In the next year, plant the seeds of the selected varieties by plant rows.

[0128] Harvest the M3 seeds obtained from M2 planted in the previous year in June of the third year. For the M3 seeds, after continuing to classify according to the first trait index, randomly take more than 100 seeds from each and stain them with 1% concentration of I2-KI. The whole shriveled grain will show blue-black within 1 minute, while the smooth and plump seeds are not stained. Thus, the whole I2-KI stained grain showing blue-black is confirmed as the second specific trait index for the starch branching enzyme SBE base mutation to produce high amylose starch, i.e., resistant starch. The lines with the whole I2-KI stained grain showing blue-black are determined as the lines to be selected for planting in the next year, and the lines not stained with I2-KI are planted as controls.

[0129] Harvest the M4 seeds obtained from M3 planted in the previous year in June of the fourth year. For the M4 seeds harvested by plant lines, after continuing to classify according to the first and second trait indexes, take 1 kg from each for the detection of quality traits. Unexpectedly, it is found that for the lines meeting the first and second specific trait indexes, the dough water absorption rate measured by the farinograph significantly increases to about 75 ml, and many lines even exceed 80 ml; while for the seeds of the control lines harvested in the same period, their dough water absorption rate is still about 60 ml, the same as that of common wheat varieties. Thus, it is determined that the high dough water absorption rate may be the third specific trait index for the starch branching enzyme SBE base mutation to produce resistant starch.

[0130] Harvest the M5 seeds obtained from M4 planted in the previous year in June of the fifth year. Observe the morphological characteristics of starch granules with a scanning electron microscope and find that the starch granules of the selected line A meeting the above 3 specific traits are irregular in shape, and some starch granules are elongated and twisted, showing long strip or crescent shape, i.e., sickle shape. The starch granules of the control line A harvested in the same period are regular, showing disc shape and oval shape. Thus, it is determined that the degree of change of A starch granules from regular oval shape to irregular crescent shape is the fourth specific trait index for the starch branching enzyme SBE base mutation to produce resistant starch.

[0131] To verify the accuracy of the aforementioned four specific trait selections, the amylose content and resistant starch content of the M6 seeds of each selected strain harvested in June of the 6th year were detected. The amylose content increased from 25% of normal wheat varieties to around 60%, and the resistant starch content increased from around 1% of normal wheat varieties to around 10%. Among them, the amylose content of Shanong RS1 with the highest content was around 65%, the resistant starch content was 11.8%, and the glycemic index GI value was 47, which was a high-resistant starch wheat variety leading at home and abroad.

[0132] Thus, it was confirmed that the cultivation of the resistant starch wheat variety was successful.

[0133] The present invention first regarded the wrinkled and dull variation of the seed coat as the first specific trait for starch branching enzyme SBE base mutation and selection of resistant starch. This is because there are many random point mutations caused by EMS treatment, and not all mutagenized varieties show wrinkled seed coat variation. In addition, most of the previous EMS mutagenesis breeding focused on mutations of good agronomic and quality traits and did not pay attention to the wrinkled seed coat variation or regarded it as a poor mutation and eliminated it. Based on the purpose of selecting special starch mutants, the present invention attached importance to the trait of epidermal wrinkling and dullness and first used it as an important index for the selection of high amylose (resistant) starch.

[0134] The present invention first regarded that the whole wrinkled grain stained blue-black within 1 minute by I2-KI staining, while the smooth and plump seeds were not stained, as the second specific trait for starch branching enzyme SBE base mutation and selection of resistant starch. This is because the previous method of using half-grain I2-KI staining of the seed section was for breeding waxy wheat varieties with mutated branching amylases. Because only the seeds with all three genes of amylose synthase (GBSS) mutated, that is, the whole waxy wheat seeds showed purplish-red on the section. Because as long as there is a small amount of amylose present, the seed section stained by I2-KI shows blue. Therefore, the I2-KI staining method has never been used for the selection of high amylose (resistant) starch varieties, and thus there has never been a report of the whole wheat grain stained blue-black by I2-KI staining by predecessors.

[0135] The present invention first regarded the significantly increased dough water absorption rate compared with common wheat as the third specific trait for starch branching enzyme SBE base mutation and selection of resistant starch. This is because those skilled in the art already know that the dough water absorption rate of common wheat with a straight-chain to branched-chain starch ratio of 25:75 is generally around 60 ml, and the water absorption rate of waxy wheat with a high branched-chain starch content is as high as around 70 ml. According to this inference, the water absorption rate of high amylose starch varieties should be low, but this study found that the water absorption rate of high amylose starch varieties was as high as around 80%. At present, there are few high amylose (resistant) starch wheat varieties bred at home and abroad, so predecessors have not reported the phenomenon of significantly increased water absorption rate of high amylose starch varieties (resistant starch wheat), let alone regarded this as an important index for the selection of high amylose starch and resistant starch wheat.

[0136] For the first time in the present invention, the irregular shape of A starch granules observed by scanning electron microscopy, with some starch granules elongated and distorted, showing a long strip or crescent shape, i.e., sickle shape, is used as the fourth specific trait for base mutation of starch branching enzyme SBE and selection of resistant starch, because gene mutation will definitely affect the change of the morphological structure of starch granules.

[0137] Based on the specific traits newly discovered in different generations above, which may be related to the mutation of starch synthase, a technical route for rapidly cultivating resistant starch wheat with the characteristics of "seed coat shrinkage + whole grain stained blue-black with I2-KI + significantly increased water absorption rate + A starch granules showing a long strip or crescent shape, i.e., sickle-shaped variation" is formed, solving the technical problems of a large number of random mutations generated by EMS mutagenesis, which require a lot of work and time for identification and recognition, and the scientific problem of the difficult and long process of cultivating high-resistant starch wheat varieties. Based on this method, after more than 10 years of exploration, it has been verified and matured. The new resistant starch line Shannong RS1 selected rapidly by this technology has an amylose content of about 60%, which is twice as high as the amylose content (31.37%) of the only reported high-amylose wheat variety Xinnong 836 in the country at present. The resistant starch content is 11.8%, and the glycemic index GI value is 47, which is a high-resistant starch wheat variety leading in the world.

[0138] Combined with the following several examples of resistant starch breeding, the technical requirements of the present invention are further described to inspire crop breeders to use the technology of the present invention to breed more and better new varieties of resistant starch (high-amylose) wheat. It should be noted that the present invention is not limited to the selection based on EMS mutagenesis materials mentioned in the claims (Example 1), and is also suitable for the selection of new varieties of high-amylose wheat (high-resistant wheat) using conventional hybrid materials of two parents (Example 2).

[0139] Those skilled in the art will understand that the following examples are only for illustrating the present disclosure and should not be regarded as limiting the scope of the present disclosure. For the technical or conditions not specified in the examples, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments not indicated by the manufacturer can be obtained as conventional products through commercial purchase.

[0140] Example 1 Rapid Breeding of the New High-Resistant Starch Variety Shannong RS1

[0141] 1. Test Materials

[0142] PH82-2-2 (distant bloodline), Shannong 20 (distant bloodline), Sankecun (local variety), GB16 (German germplasm), GB25 (Italian germplasm), and Mo 99 (CIMMYT germplasm), a total of 6, containing distant bloodlines, local germplasms, and foreign germplasms. Among them, PH82-1-2-2 and Shannong 20 are varieties with distant bloodlines cultivated by Shandong Agricultural University, and the other 4 are provided by the Crop Research Institute of the Chinese Academy of Agricultural Sciences.

[0143] 2. Material Treatment and Planting

[0144] (1) Seed EMS Treatment:

[0145] The EMS (ethyl methanesulfonate) mutagen was provided by Beijing Solarbio Science & Technology Co., Ltd. 1000 seeds of each of the 6 test materials were selected and pre-soaked in distilled water for 8 hours, and then placed in 0.8 mol / L EMS solution for 16 hours of mutagenesis treatment, with at least 3 shakes during this period. When the soaking treatment was completed, the mutagenized wheat grains were repeatedly rinsed with clean water, left to dry for one day, and then sown by strain.

[0146] (2) Material Planting

[0147] The mutagenized seeds were sown by strain in the breeding experimental field of Shandong Agricultural University with good water and fertilizer conditions. Each variety was sown in 10 rows, with 100 seeds in each row. At the seedling emergence stage, the seedling emergence potential, emergence rate, and seedling growth status of each mutagenized variety were recorded, and water, fertilizer, disease, and pest management were carried out according to the normal planting requirements of wheat until harvest. The planting and field management of M1 - M5 generation materials were basically the same as those of M1, except that sowing and harvesting were carried out by individual plants or plant lines according to the requirements of different generations.

[0148] 3. Measurement Items and Methods

[0149] (1) Whole Seed I2-KI Staining Identification

[0150] Prepare a petri dish with a diameter of 10 cm, put 100 - 200 seeds to be measured, and make them evenly distributed in a single layer; add an appropriate amount of I2-KI solution, and stir with a glass rod if necessary to completely immerse the seeds in the solution. After 1 minute, count the staining situation of the grains.

[0151] (2) Dough Water Absorption Measurement

[0152] Adopt GB / T 14614—2019 "Inspection of Grain and Oil - Method for Testing the Rheological Properties of Wheat Flour Dough by Farinograph", and use an 810130 electronic farinograph for measurement. Dough water absorption is an important farinograph parameter of the rheological properties of wheat flour dough and is directly related to food processing quality.

[0153] (3) Amylose Content Measurement

[0154] Determined according to the amylose determination method of GB / T 15683-2008, and the measuring instrument is a full-automatic amylose analyzer produced by Kopman, France.

[0155] (4) Determination of resistant starch content

[0156] According to the resistant starch content determination method of T / CI 005—2022, α-amylase and amyloglucosidase (AMG) were used to incubate in a shaking water bath at 37 °C for 16 hours to dissolve and hydrolyze non-resistant starch into D-glucose. Then, the reaction was terminated by adding ethanol or industrial methylated spirit (IMS), and resistant starch (RS) was obtained by centrifugation. Finally, the content of D-glucose was determined by GOPOD reagent, and thus the content of resistant starch was indirectly determined. The measuring instrument is a UV-visible spectrophotometer produced by Shanghai Yuanxi.

[0157] 4. Results and analysis

[0158] (1) Specific mutations and identification of seed coat appearance

[0159] The harvested M1 seeds were taken back to the laboratory, with the original parent before induction as the control, and the variation of grain appearance traits was mainly observed. It was found that about 10% of the seeds of PH82-1-2-2 M1 had special variant grains with shrunken and dull seed coats, while the appearance of grains of other varieties changed insignificantly ( Figure 2 、 3 ). Therefore, PH82-1-2-2 was retained as the key combination, and the seeds were divided into two categories: seeds with appearance variation and seeds without variation, and were sown separately.

[0160] The harvested M2 seeds were carefully investigated and identified by single ear and single plant. The results showed that 90% of the offspring of the seeds with shrunken and dull seed coats of PH82-1-2-2 still maintained the traits of shrunken and dull, while the types with smooth and dull seed coats and other tested seeds did not show shrunken seed coats ( Figure 4 ). Therefore, the stably inherited trait of shrunken and dull seed coat was initially set as the first trait marker for possible mutations in the base of starch branching enzyme (SBE). And 30 plant lines were planted according to the two types of this trait marker.

[0161] (2) Specific mutations and identification by I2-KI staining

[0162] The M2 generation seeds were harvested according to different types of plant lines. In order to exclude the possibility that EMS mutagenesis may also cause base mutations in granule-bound starch synthase (GBSS) and produce waxy wheat types, the seeds were cut with a blade according to the conventional method and soaked in I2-KI staining. The cross-section of whole waxy wheat grains showed purple-red ( Figure 5A ), and the cross-section of normal common wheat seeds showed blue ( Figure 5B), proving that these strains are not waxy wheat produced by mutation, while the whole grains of high amylose wheat seeds are bluish-black( Figure 6 ), thus determining that the whole grain showing bluish-black by I2-KI staining is the second trait marker.

[0163] (3) Specific mutation and identification of dough water absorption rate

[0164] Harvest the M3 generation seeds according to different types of plant lines and conduct tests on conventional quality traits. It is found that there are little variations in grain protein content, gluten content, etc. between the two mutant type plant lines. However, at the first step of the farinograph measurement of dough rheological properties, extremely significant differences in water absorption rates are found between the two types. The water absorption rate of the mutant type with shrunken and dull epidermis is as high as about 80 ml, while that of the type with plump and shiny epidermis is only about 60 ml, the same as the original variety( Figure 7 、 8 ). Thus, it is further determined that the high dough water absorption rate is the third trait marker for the mutation of the base of starch branching enzyme (SBE) to produce high amylose (resistant starch).

[0165] (4) Scanning electron microscopy observation and identification of the morphological characteristics of M5 seed starch granules

[0166] Observed by a CEM3000 desktop scanning electron microscope (SEM), it is found that the A starch granules of the selected plant lines meeting the above 3 specific traits are irregular in shape, and some starch granules are elongated and distorted, showing a long strip or crescent shape, that is, sickle-shaped( Figure 9 ). The A starch granules of the control plant lines harvested during the same period are regular, showing a disc shape and oval shape( Figure 10 ). Thus, it is determined that the degree of the change of A starch granules from regular oval shape to irregular crescent shape is the fourth specific trait index for the mutation of the base of starch branching enzyme SBE to produce resistant starch.

[0167] (5) Identification of amylose content and resistant starch content of specific mutant plant lines

[0168] Take the mutants with "shrunken and dull grains + I2-KI staining of grain cross-section showing bluish-black + high water absorption rate + obvious changes in the configuration of some starch granules", and conduct high amylose determination according to the national standard method. It is measured that the content of amylose in total starch is increased to about 60% (the high value of high amylose wheat varieties reported at home and abroad currently), while that of the type with plump and shiny epidermis is only about 25%, the same as the original variety. Thus, it is determined that the mutants with "shrunken and dull grains + high water absorption rate + I2-KI staining of grain cross-section showing bluish-black + obvious changes in the configuration of some starch granules" are new high amylose wheat varieties.

[0169] The obtained new high amylose mutants such as Shannong RS1, a new high amylose strain, were further determined for the resistant starch content according to the method for determining the resistant starch content in T / CI 005—2022. It was found that the resistant starch of the new high amylose variety increased from less than 1% of the original variety to about 11.8% (the high value of the reported high resistant starch wheat varieties at home and abroad). Thus, it was determined that the new high amylose wheat variety was also a new high resistant starch wheat variety, and the ratio of amylose to resistant starch was about 5:1.

[0170] Based on EMS mutagenesis, the technique of "shrunken and dull epidermis + high water absorption rate + blue-black staining of grains with I2-KI + determination of amylose and resistant starch + obvious changes in the configuration of some starch granules" is a rapid and effective method for quickly selecting and breeding new varieties with high amylose and resistant starch contents. From EMS mutagenesis to the detection and identification of high resistant starch and high amylose, that is, the new high resistant starch variety Shannong RS1 was cultivated. The contents of high resistant starch and high amylose in Shannong RS1 were as high as about 11.8% and about 65% respectively (the detection results are as Figure 11 、 12 shown), both of which were at the leading levels at home and abroad.

[0171] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0172] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A breeding method for a new variety of wheat with high amylose content and high resistant starch content, characterized in that, Including: S1: Using EMS to mutagenize parental wheat seeds, planting the mutagenized seeds M0, and obtaining M1 seeds harvested by plant line; S2: Screening the plant lines to which the M1 seeds with shrunken and dull seed coats belong, and planting them to obtain M2 seeds harvested by plant line; S3: Screening the plant lines to which at least 90% of the M2 seeds with shrunken and dull seed coats belong, and planting them to obtain M3 seeds harvested by plant line; S4: Staining the M3 seeds with I2-KI, screening the plant lines to which the grains that are entirely stained blue-black belong, and detecting the amylose and resistant starch contents of at least a part of the seeds of this plant line to determine the plant lines with amylose content reaching at least 45% and / or resistant starch content reaching at least 6%, so as to obtain a new variety of wheat with high amylose and high resistant starch.

2. The breeding method according to claim 1, characterized in that, The breeding method further includes: S5: Continuing to plant the plant lines to which the M3 seeds with whole grains stained blue-black by I2-KI belong for at least 1 generation, detecting the amylose and resistant starch contents of the seeds harvested by plant line, and determining the plant lines with amylose content reaching at least 45% and / or resistant starch content reaching at least 6%, so as to obtain a new variety of wheat with high amylose and high resistant starch.

3. The breeding method according to claim 1, characterized in that, The breeding method further includes: S6: Continuing to plant the plant lines to which the M3 seeds with whole grains stained blue-black by I2-KI belong to obtain M4 seeds harvested by plant line; S7: Detecting the dough water absorption rate of the M4 seeds harvested by plant line, screening the plant lines to which the grains with dough water absorption rate reaching at least 75 mL belong, which are the new variety of wheat with high amylose and high resistant starch, Or, planting the plant lines to which the grains with dough water absorption rate reaching at least 75 mL belong for at least 1 generation, so as to obtain a new variety of wheat with high amylose and high resistant starch.

4. The breeding method according to claim 1, characterized in that The breeding method further includes: S8: Continuing to plant the plant lines to which the M3 seeds with whole grains stained blue-black by I2-KI belong to obtain M4 seeds harvested by plant line; S9: Detecting the dough water absorption rate of the M4 seeds harvested by plant line, screening the plant lines to which the grains with dough water absorption rate reaching at least 75 mL belong and continuing to plant them to obtain M5 seeds harvested by plant line; S10: Conducting morphological identification of A starch granules on the M5 seeds harvested by plant line, screening the plant lines to which the seeds with partially irregularly mutated A starch granule shapes belong, which are the new variety of wheat with high amylose and high resistant starch, Or, planting the plant lines to which the seeds with irregular A starch granule shapes belong for at least 1 generation, so as to obtain a new variety of wheat with high amylose and high resistant starch.

5. The breeding method according to claim 4, characterized in that, The breeding method further includes, in step S10, after planting the plant lines to which the seeds with irregular A starch granule shapes belong for at least 1 generation, detecting the amylose and resistant starch contents of the seeds harvested by plant line, and determining the plant lines with amylose content reaching at least 45% and / or resistant starch content reaching at least 6%, so as to obtain a new variety of wheat with high amylose and high resistant starch.

6. The breeding method according to claim 1, characterized in that When conducting the mutagenesis treatment, the concentration of EMS is 0.5 - 1.0 mol / L; Optionally, the time of the mutagenesis treatment is 8 - 24 h.

7. The breeding method according to claim 1, characterized in that, The parental wheat seeds are derived from wheat varieties with distant bloodlines; Optionally, the wheat varieties with distant bloodlines include PH82 - 2 - 2 and Shannong 20.

8. The breeding method according to claim 7, characterized in that, There are at least 1000 parental wheat seeds, preferably 1000 - 10000 seeds.

9. A wheat, characterized in that, It is obtained by incrementally stacking through the breeding method described in any one of claims 1 - 8.

10. The wheat according to claim 9, characterized in that, The wheat is Shannong RS1, and the preservation number is CGMCC NO.: 30792.

11. Use of the wheat obtained by the breeding method described in any one of claims 1 - 8 in the preparation of starch products.

12. A starch product, characterized in that, The starch product is prepared from wheat with a straight-chain starch content of at least 45% and a resistant starch content of at least 6%.

13. The starch product according to claim 12, wherein The starch product is prepared from wheat obtained by incrementally stacking through the breeding method described in any one of claims 1 - 8.

14. A method for preparing a starch product, characterized in that, Including: A. Cultivate the wheat variety obtained by the breeding method described in any one of claims 1 - 8 and harvest the seeds; B. Process the seeds to obtain starch products.

15. A method for cultivating a new variety of wheat with high amylose and high resistant starch, characterized in that, It is obtained by at least one of the following methods: (1) Use the wheat variety obtained by the breeding method described in any one of claims 1 - 8 as a parent for cultivation; (2) Gene editing; (3) Cross breeding.

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