Rapid breeding method of waxy wheat

Abstract

The invention relates to the field of wheat breeding, in particular to a rapid breeding method of waxy wheat. The invention provides a rapid breeding method of waxy wheat, which combines a plurality of experimental techniques such as tissue culture, KASP molecular marker and iodine staining identification, and can rapidly create a new line of waxy wheat. According to the method, the breeding time can be effectively and remarkably shortened, and a new method is provided for wheat variety improvement.

Description

Technical Field

[0001] The invention relates to the field of wheat breeding, in particular to a rapid breeding method for waxy wheat. Background Art

[0002] With the diversification of people's eating habits, glutinous food has gradually gained people's favor due to its unique food characteristics. The breeding of glutinous wheat varieties and its application in the food processing industry have gradually become one of the focuses of social attention. Waxy wheat has a specific starch composition and unique flour processing quality. It is particularly suitable for making noodles and other pasta and as a raw material for brewing. It is a high-quality special wheat type with broad application prospects. The biggest difference between glutinous wheat flour and ordinary wheat flour lies in the gelatinization and retrogradation characteristics of starch. The starch gelatinization temperature and DSC determination starting temperature of glutinous wheat flour are relatively low, the water holding capacity and swelling capacity are strong, the degree of retrogradation and aging is small, the freeze-thaw stability is good, the starch transparency, swelling potential and peak viscosity are lower than potatoes, cassava, glutinous corn, etc., and it can be widely used in the development of specialty foods and the production of refrigerated and quick-frozen foods. For example, adding glutinous wheat flour to wheat flour can improve the quality and taste of quick-frozen products. The gelatinization and retrogradation of glutinous wheat starch are the main factors affecting high-moisture traditional oriental foods. Its special starch composition plays an important role in noodle products and starch processing. The low amylose content of glutinous wheat helps to improve the quality of bread and extend the shelf life of bread. The steamed bread with a proper amount of glutinous wheat flour has a better flavor and can be developed as a new product. 25.0% glutinous wheat flour can make steamed bread have a better texture and the sensory score reaches the maximum, while adding 8% glutinous wheat flour can make steamed bread products with good taste and storage performance. Glutinous wheat flour can also be used to make specialty pastries and can be used as an additive in food packaging paper, concentrates, pastes and environmentally friendly plastics. Adding glutinous wheat flour to ordinary wheat flour can improve the sensory and texture characteristics of noodles. It can be seen that glutinous wheat can improve the processing quality of special foods, increase the yield and quality of wine, improve the preservation ability and extend the shelf life, and has many advantages, and has broad application prospects. Therefore, conducting research on the rapid and efficient breeding of new glutinous wheat lines is of great significance for shortening the breeding process, improving breeding efficiency, and promoting the efficient breeding of new high-quality special wheat varieties.

[0003] Traditional hybrid breeding combines parents according to breeding goals, and breeds varieties through continuous generation selection. This method is mainly based on conventional phenotypic screening, which has disadvantages such as long cycle and high cost. Wheat is a hexaploid species, containing three subgenomes A, B, and D. To achieve gene aggregation of breeding target trait genes, it takes a longer cycle than diploid crops. Therefore, it is necessary to improve traditional breeding methods to obtain a waxy wheat. Summary of the invention

[0004] The purpose of the present invention is to provide a rapid breeding method for waxy wheat to solve the problems existing in the above-mentioned prior art. The breeding method provided by the present invention can effectively and significantly shorten the breeding time.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a rapid breeding method for waxy wheat, comprising the following steps:

[0007] (1) Using Shumai 133 as the female parent and low-amylose wheat as the male parent for hybridization to obtain F1 generation seeds;

[0008] (2) sowing the F1 generation seeds, taking Shumai 133 as the recurrent parent, and performing backcrossing. After 15-21 days of pollination, the young embryos of the backcrossed plants were collected for tissue culture to obtain BC1F1 generation seeds;

[0009] (3) sowing the BC1F1 generation seeds, identifying the pollen of the BC1F1 plants by pollen iodine staining, selecting the BC1F1 plants with reddish brown pollen, and performing backcrossing with Shumai 133 as the recurrent parent. After 15-21 days of pollination, the young embryos of the backcrossed plants were collected for tissue culture to obtain BC2F1 generation seeds;

[0010] (4) Repeat step (3) once to obtain BC3F1 generation seeds;

[0011] (5) sowing the BC3F1 generation seeds, identifying the pollen of the BC3F1 plants by pollen iodine staining, selecting BC3F1 plants with reddish-brown pollen for self-pollination, and taking young embryos from the self-pollinated plants for tissue culture 15-21 days after pollination to obtain BC3F2 seeds;

[0012] (6) sowing the BC3F2 seeds, conducting molecular tracing of the Wx-A1 gene and the Wx-D1 gene, and then self-pollinating to obtain BC3F3 seeds homozygous for waxy wheat;

[0013] (7) Repeat step (6) three times to obtain the glutinous wheat.

[0014] Preferably, in steps (2) to (5), the method for preparing the culture medium used for tissue culture comprises the following steps:

[0015] Mix Ms culture medium base salt, casein hydrolyzate, maltose and water in a mass volume ratio of 2.15 g:0.5 g:15 g:100 mL, and adjust the volume to 250 mL to obtain a mixture 1;

[0016] 1.75 g of plant gel and water were mixed and the volume was adjusted to 250 mL to obtain mixture 2;

[0017] The medium was obtained by mixing 250 mL of mixture 1, 250 mL of mixture 2, 125 μL of IBA, 62.5 μL of NAA, and 2.5 mL of vitamins.

[0018] Preferably, in steps (2)-(5), the tissue culture time is 15-20 days, the temperature is 25° C., the illumination time is 16 hours, the illumination intensity is 3000-5000 Lux, and the humidity is 40%.

[0019] Preferably, the molecular tracking of the Wx-A1 gene includes the following steps: using the wheat genome to be tested as the template DNA, amplifying using an upstream primer with a nucleotide sequence such as that shown in SEQ ID NO.1-2 and a downstream primer with a nucleotide sequence such as that shown in SEQ ID NO.3, and performing fluorescence scanning on the amplified product; if the fluorescence scanning obtains a HEX fluorescence signal, it is determined that the wheat to be tested is a wheat variety lacking the Wx-A1 gene.

[0020] Preferably, the molecular tracking of the Wx-D1 gene includes the following steps: using the wheat genome to be tested as the template DNA, amplifying using upstream primers with nucleotide sequences such as those shown in SEQ ID NO.4-5 and downstream primers with nucleotide sequences such as those shown in SEQ ID NO.6, and performing fluorescence scanning on the amplified products. If the fluorescence scanning obtains a HEX fluorescence signal, it is determined that the wheat to be tested is a wheat variety lacking the Wx-D1 gene.

[0021] Preferably, the low-amylose wheat includes Sichuan glutinous wheat 1456.

[0022] The present invention provides a primer set for identifying waxy wheat, the primer set comprising a first primer set and a second primer set;

[0023] The first primer set includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.1-2 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.3;

[0024] The second primer set includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.4-5 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.6.

[0025] The present invention provides the use of the primer set in identifying waxy wheat varieties and / or breeding waxy wheat varieties.

[0026] The present invention discloses the following technical effects:

[0027] The present invention combines tissue culture, KASP molecular markers and iodine staining identification with multiple experimental techniques, identifies through SDS-PAGE biochemical markers and starch phenotypes, quickly creates new waxy wheat strains, and analyzes the agronomic traits, starch characteristics and processing quality of the new waxy wheat strains, in order to provide methods and germplasm for wheat quality improvement. In the present invention, tissue culture, KASP molecular markers and iodine staining identification are used in combination to make breeding faster and more convenient. Wheat is rapidly multiplied by using the rapid breeding method of tissue culture, and embryos are selected for the materials that are strong 15-21 days after cross pollination. After embryo selection, the seedlings emerge after 15-20 days of cultivation in a 25°C incubator, and are planted in a greenhouse until maturity. A season is 90 days in total, which saves half the cycle compared to conventional field planting. Therefore, the application of tissue culture technology greatly shortens the planting cycle and improves breeding efficiency. KASP molecular markers can screen the target traits in the population at the seedling stage, leaving only plants containing the target traits, greatly reducing breeding costs. In the continuous backcrossing process of the present invention, accurately selecting the heterozygous type single plant of the Wx-1 locus is a key link in shortening the material creation cycle. The iodine staining method is used to stain and identify the pollen of a single plant, and the heterozygous single plant can be determined according to the proportion of glutinous pollen, which is convenient and fast, and can significantly improve the accuracy of genotype identification and improve hybridization efficiency. In a random group, the identification results of a single plant and a single grain are consistent in theory, but the workload of single grain identification is relatively small. At the same time, combined with iodine staining of mature grains, the starch composition of the grains can be quickly and efficiently identified, thereby improving the efficiency of phenotypic identification. The present invention combines conventional hybridization with a variety of breeding methods and techniques such as molecular marker-assisted selection, tissue culture generation, starch iodine staining identification, and off-site generation, integrates innovative rapid breeding technology, efficiently creates a new waxy wheat line, realizes 8 generations of breeding in 2 years, and the breeding efficiency is more than 1 times higher than that of traditional breeding methods.

[0028] In addition, the present invention uses the rolling backcrossing method to successfully transfer the genes Wx-A1 and Wx-D1 that control the waxy trait of wheat grains into some excellent parents at the same time. While obtaining the glutinous genes of the donor parents, the excellent traits of the parents, such as high yield, lodging resistance, stress resistance, etc., are inherited, and disease resistance is also improved, indicating that the waxy gene mutation itself does not have an adverse effect on other excellent traits such as yield.

[0029] At the same time, the present invention also has the following advantages:

[0030] 1. The present invention selects Shumai 133 (Wx-AAbbDD) as the recipient parent and Chuannuomai 1456 (Wx-aabbdd) as the Wx gene-deficient donor parent to prepare a hybrid combination, combines plant tissue culture and greenhouse generation, and quickly and efficiently creates a new glutinous wheat material. The F1 generation is hybridized in a greenhouse, and the F1 generation embryos are used as explants for tissue culture, and the generations are rapidly increased in the greenhouse. The hybrid offspring is backcrossed with Shumai 133 three times, and all subsequent backcross offspring (BC1F1-BC3F1) are tissue cultured and generated by the same method. After self-pollination five times, the BC3F5 generation strain is obtained.

[0031] 2. The pollen and KASP molecular markers and iodine staining of grain sections were used to detect the backcross and self-pollinated progeny, and the SDS-PAGE method was used for identification, which greatly improved the selection efficiency. The pollen iodine staining method was used to detect the backcross progeny, and the heterozygous plants with 1 / 4 glutinous pollen (reddish brown) were selected in each generation, and then backcrossed with the recurrent parent Shumai 133. Since the Wx-B1 locus of Shumai 133 itself is a natural deletion type, two gene-specific KASP markers of Wx-A1 gene and Wx-D1 gene were developed according to the Wx gene sequence. The above KASP markers were used to type and identify the heterozygous types of 217 BC3F2 generations, and 29 strains were screened out for recessive homozygous mutations in the two gene loci of Wx-A1 and Wx-D1. The Waxy protein deficiency was further detected and verified by iodine staining of grain sections and SDS-PAGE. The results showed that none of the 29 strains expressed Waxy protein. The 29 lines were self-pollinated continuously, and finally a new waxy wheat line with Wx-1 gene locus silencing material (Wx-aabbdd) and stable agronomic traits was obtained. This method greatly improved the breeding efficiency and shortened the breeding time by 3-4 years compared with traditional breeding methods.

[0032] 3. Agronomic trait investigation shows that the new glutinous wheat strain created has introduced the glutinous Wx-1 gene and inherited the excellent agronomic traits of the parents. Compared with the excellent parent Shumai 133, there is no obvious difference in the main agronomic traits of the BC3F5 generation strains, such as grain length, surface area, 100-grain weight, plant height, flag leaf length, spikelet number, and tiller number. The present invention uses the rolling backcrossing method to successfully transfer three pairs of genes Wx-A1, Wx-B1, and Wx-D1 that control the full glutinous traits of wheat grains to excellent parents at the same time. While obtaining the glutinous genes of the donor parents, it also inherits the excellent traits of the parents, such as high yield, lodging resistance, and stress resistance, indicating that the introduction of the Waxy gene mutation itself does not have an adverse effect on other excellent traits such as yield.

[0033] 4. Comprehensive analysis of the quality indicators of the new glutinous wheat materials, such as total starch content, amylose content, sedimentation value, RVA parameters and powder parameters, showed that the new glutinous wheat strain had unique starch composition and physicochemical properties. The results of starch composition determination showed that the amylose content of the created new material varied from 0-10%, and the amylopectin content was above 90%, which met the starch composition requirements in the Sichuan Province Waxy Wheat Variety Approval Standard. Analysis of the microstructure of glutinous wheat starch showed that the starch granule morphology and the average degree of polymerization of starch chain length were not significantly different from those of Shumai 133, but the starch branching degree increased. Although the starch crystallinity of glutinous wheat increased significantly, the type of crystal structure did not change. In terms of physicochemical properties, the gelatinization peak temperature, termination temperature and thermal enthalpy value of glutinous wheat were extremely significantly reduced. The determination of quality parameters showed that the sedimentation value of the new glutinous wheat strain was reduced, the RVA gelatinization time was reduced, and the powder parameter stability time was reduced. Therefore, the glutinous wheat formed by the loss of the Waxy subunit has a significant change in starch composition and properties due to the significant reduction in amylose content, giving it unique processing quality characteristics and uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 is an experimental flow chart; wherein WN15 (Wx Null-15) in the figure is the abbreviation of Sichuan glutinous wheat 1456;

[0036] Figure 2 This is a greenhouse hybridization diagram of Shumai 133 and Chuannuomai 1456, where a and b are the female parents of Shumai 133 in the greenhouse, and cd are the fruits of Chuannuomai 1456 after pollination in the greenhouse;

[0037] Figure 3 This is the tissue culture of backcross materials, where a is the process of picking embryos on a clean bench; b is the rooting culture process; and c is the greenhouse planting state;

[0038] Figure 4 Pollen iodine staining identification of different backcross generations; WN15 (Wx Null-15) in the figure is the abbreviation of Chuannuomai 1456;

[0039] Figure 5 This is the KASP molecular marker genotyping; blue is Wx recessive homozygous, yellow is Wx dominant homozygous, and green is Wx heterozygous;

[0040] Figure 6This is the KASP molecular marker genotyping verification of the self-fertilized material; blue is Wx recessive homozygous, yellow is Wx dominant homozygous, and green is Wx heterozygous;

[0041] Figure 7 This is the iodine staining verification of self-pollinated material grains; a and b are the iodine staining results of BC3F5 generation grain slices, which show heterozygous grains; c is the iodine staining results of BC3F5 generation grain slices, which show homozygous waxy wheat; blue is non-waxy wheat, and reddish brown is waxy wheat;

[0042] Figure 8 The SDS-PAGE results of Shumai 133 and part of SM133 BC3F5; wherein, M is a marker, SM133 is Shumai 133, and lanes 1-8 are SM133 BC3F5;

[0043] Fig. 9 The field performance of Shumai 133 and some SM133 BC3F5 lines;

[0044] Fig.10 The grain morphology of Shumai 133 and part of SM133BC3F5;

[0045] Fig.11 Comparison of starch granule size between Shumai 133 and SM133-Wx-null-54 materials;

[0046] Fig.12 Comparative analysis of starch structure between Shumai 133 and SM133-Wx-null-54; a and b are the starch granule structures of Shumai 133; c and d are the starch granule structures of SM133-Wx-null-54; e and f are the starch granule structures of Shumai 133 grain slices; g and h are the starch granule structures of SM133-Wx-null-54 grain slices;

[0047] Fig.13 It is a comparative analysis of the chain length distribution of Shumai 133 and SM133-Wx-null-54 materials;

[0048] Fig.14 This is a comparative analysis of the starch branching degree of Shumai 133 and SM133-Wx-null materials; the horizontal axis represents the chemical shift (ppm); the vertical axis represents the absorption intensity;

[0049] Fig.15 It is a comparative analysis of XRD diffraction between Shumai 133 and SM133-Wx-null-54 materials;

[0050] Fig.16 This is the RVA viscosity analysis of Shumai 133 and some SM133 BC3F5 strains;

[0051] Fig.17 This paper is a comparative analysis of powder quality parameters of Shumai 133 and SM133 BC3F5 lines. DETAILED DESCRIPTION

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0054] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0055] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0056] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0057] Example 1

[0058] 1. Materials and methods

[0059] 1.1. Parent material selection

[0060] The wheat materials used in this example include: wheat varieties Shumai 133 (SM133) and Chuannuomai 1456 (named as glutinous wheat germplasm Wx Null-15, abbreviated as Wx Null-15 or WN15 in the experiment), all of which were provided by the Wheat Research Institute of Sichuan Agricultural University. The technical route is as follows Figure 1 shown.

[0061] Shumai 133 is a new national wheat variety selected by the Wheat Research Institute of Sichuan Agricultural University. It has outstanding advantages such as disease resistance, high yield, frost resistance, and ear germination resistance. It is suitable for planting in the flat or hilly areas of the upper reaches of the Yangtze River and has been selected as the main agricultural variety in Sichuan Province in 2023. In the early stage, this research group used Shumai 133 (Wx-AAbbDD) as the recurrent parent and Chuannuomai 1456 (Wx-aabbdd) as the donor parent. By comprehensively utilizing rolling backcrossing combined with pollen iodine staining, KASP molecular marker-assisted breeding, slice iodine staining and SDS-PAGE, the waxy wheat with Shumai 133 as the genetic background was successfully screened in BC3F2, and continued to self-pollinate to BC3F5. The field test was planted in plots, with 10 rows per plot, repeated 3 times, a row length of 1.5m, and a row spacing of 0.3m. It was completed in the greenhouse of Sichuan Agricultural University, the Wenjiang Experimental Base in Chengdu, Sichuan Province, and the summer breeding base in Kangding, Sichuan Province.

[0062] 1.2. Requirements for pollen of female and male parents of hybridization

[0063] 1.2.1、Maternal parent selection requirements:

[0064] (1) Selecting ears: Before emasculating the mother plant, select healthy, disease-free main stem ears or large tillering ears; (2) Preparing ears: Use tweezers to remove the spikelets at the top and base of the ear, keep the spikelets in the middle of the ear, and use scissors to cut off the upper third of the wheat husk; (3) Emerging tassels: The lemma and outer lemma of the wheat are tightly closed. In order to remove the anthers in the flower, use tweezers to gently separate the lemma and outer lemma, and then use tweezers to remove the anthers.

[0065] 1.2.2 Requirements for collecting male parent pollen:

[0066] (1) Flowering: It is carried out in the morning when the wheat is in full bloom. If 1-2 florets have opened in the male wheat ear, it means that a large number of stamens will soon open. In order to quickly obtain pollen, gently pull the above-mentioned ears of wheat that have already opened from bottom to top 2-3 times with your hands, and pull several ears at the same time. After a while, you can see that the inner and outer lemmas of a large number of florets have stretched out, revealing pale yellow anthers. (2): Pollination: When the stigma of the emasculated floret of the female wheat ear is feather-like and shiny, it means that the stigma is mature and pollination should be carried out immediately, which is usually the second day after emasculation. Therefore, pollination should be carried out in the morning of the second day after emasculation. (3) Bagging: Pollination should be carried out in order, from top to bottom, one side is pollinated and then the other side. After pollination, put the paper bag back on and write the male parent's name and pollination date on the other side of the plastic card. Then cut off a corner of the plastic card to indicate the end of pollination. Ten days after pollination, the paper bags were removed to allow the hybrid ears to grow and develop normally.

[0067] 1.3 Tissue Culture

[0068] 1.3.1 Preparation of plant regeneration medium

[0069] (1) Add 100 mL of water to a 500 mL beaker, then add 2.15 g of MS medium base salt (Ms salt), 0.5 g of casein hydrolysate (Gasein hydrolysate), and 15 g of maltose in sequence, stir evenly, and adjust the pH value to 5.8. Then use a graduated cylinder to make up to 250 mL and pour into the prepared sterile bottle.

[0070] (2) Add distilled water to 250 mL, weigh 1.75 g of Phytagel, pour into a sterile bottle, and sterilize by high pressure.

[0071] (3) Turn on the ultraviolet light of the clean bench for sterilization for 30 minutes, then mix the sterilized (1) and (2) together, add 125μL IBA (3-1ndole Butyric Acid), 62.5μL NAA (1-Naphthlcetic Acid) and 2.5mL 2000× BCI (Vitamin), shake evenly, and pour into the sterilized test tube. After the culture medium solidifies, seal it and place it on the clean bench for use.

[0072] 1.3.2 Tissue culture

[0073] (1) In a greenhouse or field, 15-21 days after pollination, wheat seeds can be taken for tissue culture after they are fruiting. The peeled seeds in the grain-filling stage are placed in a 200 mL bottle for easy cleaning.

[0074] (2) Wash with clean water 2-3 times and then place on a clean bench for operation. Clean with 75% disinfectant alcohol for 1 minute, then disinfect with sodium hypochlorite for 15-20 minutes. After disinfection, wash with sterilized distilled water 4-5 times.

[0075] (3) After cleaning, you can pick the embryos. First, use two tweezers to disinfect them under an alcohol lamp, then pour the seeds into a sterilized empty culture dish, peel off the seed coat, pick out the white embryo part, put it into the prepared regeneration medium test tube, seal it, and place the test tube in a 25℃ incubator for 15-20 days before transplanting it into the soil and cultivating it in a greenhouse.

[0076] (4) Plant regeneration culture was carried out in an incubator. The culture conditions were set at a temperature of 25°C, a light duration of 16 hours, a light intensity of 3000-5000 Lux, and a humidity of 40%. Seedling emergence was observed every five days. After 15-20 days of culture, when the young embryos differentiated into green seedlings and a strong root system, seedlings could emerge.

[0077] (5) After germination, the seedlings are transplanted into nutrient soil and cultivated in a greenhouse.

[0078] All subsequent backcross progeny (BC1F1-BC3F5) were cultured and bred in the same way.

[0079] 1.3.4. Iodine staining of wheat pollen grains

[0080] Fresh pollen from the plants of each hybrid and backcross generation in full bloom was placed on a glass slide, stained with 0.067% I2 and 0.67% KI, and observed under a microscope. Reddish brown pollen is glutinous pollen. Since the glutinous nature of wheat is controlled by three pairs of genes, Wx-A1, Wx-B1, and Wx-D1, but the Wx-B1 of both parents is of the deletion type, only the separation combination of the two loci Wx-A1 and Wx-D1 needs to be considered. In theory, about 1 / 4 of the pollen of the hybrid offspring plants of all glutinous and non-glutinous will be glutinous pollen. Therefore, plants with 1 / 4 glutinous pollen (reddish brown) are selected in each generation of rolling backcrossing, and then continuously backcrossed with the parent SM133, and then self-pollinated after 3 consecutive backcrossings.

[0081] 1.4. KASP Mark Development

[0082] 1.4.1 Primer design

[0083] The online primer design website Poly-Marker (http: / / polymarker.tgac.ac.uk, / ) was used to design and generate KASP markers. The KASP primer sequence information is shown in Table 1.

[0084] Table 1 KASP primer sequences

[0085]

[0086] 1.4.2 DNA extraction

[0087] Take fresh green leaves of individual plants of the parent and backcross material population (BC3F2-BC3F5) and put them into 2.0 centrifuge tubes (marked with numbers), put them into liquid nitrogen and quickly freeze them for later use. Use the CTAB method to extract DNA from individual plants.

[0088] 1.4.3 Reaction conditions

[0089] The genotyping of the hybrid offspring was performed using a Bio-Rad CFX96 Touch fluorescent quantitative PCR instrument (Bio-Rad, USA).

[0090] The reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0091] Table 2 Reaction system

[0092] Element Volume (μL) HiGeno2×ProbeMix 5 SNP-Specific Primers 2 Template DNA 1 <![CDATA[ddH2O]]> 2 Total 10

[0093] Table 3 Reaction procedure

[0094]

[0095] CFX Manager software (Bio-Rad, USA) was used to analyze the experimental data. FAM and HEX were used to distinguish gene loci for genotyping, and ROX was used to correct errors.

[0096] 1.5. Iodine staining of wheat grains

[0097] Select the seeds of the hybrid offspring of SM133 and Chuannuomai 1456 harvested in the field or greenhouse and perform section iodine staining to detect whether the seeds are waxy wheat. The specific operation is as follows:

[0098] (1) When slicing, slowly press the sample toward the blade and use tweezers to gently press and cut into slices.

[0099] (2) As the total slice area increases, use tweezers to gently lift the slice to prevent it from sticking to the knife surface.

[0100] (3) After the final sectioning is completed, use forceps to transfer the sections to a 0.2 centrifuge tube, add 5M urea, and simulate gelatinization for 12 hours. After 12 hours, add iodine solution (0.2% I2, 2% KI) for iodine staining observation. Blue is non-glutinous wheat, and reddish brown is glutinous wheat.

[0101] All subsequent self-pollinated progenies (BC3F1-BC3F4) were sectioned and iodine stained using the same method.

[0102] 1.6 SDS-PAGE

[0103] The SDS-PAGE method refers to the method described in "Molecular mechanism of wheat EMS mutant Wx-A1 deletion mutation and its effect on starch quality" (Luo Mi. Molecular mechanism of wheat EMS mutant Wx-A1 deletion mutation and its effect on starch quality [D]; Sichuan Agricultural University, 2019.).

[0104] 1.7 Analysis of wheat grain characteristics

[0105] The seed length, width and surface area were determined using a near-infrared analyzer DA7250 series (China). Twenty seeds of each material were placed on a near-infrared scanner plate for near-infrared scanning analysis.

[0106] 1.8. Determination of starch content and composition

[0107] 1.8.1. Determination of total starch content

[0108] The total starch content was determined according to the procedure of Megazyme Total Starch Assay Kit (K-TSTA), and the independent sample T test was used for statistical analysis.

[0109] 1.8.2 Determination of amylose content

[0110] The amylose content was determined by referring to the steps of the Megazyme Amylose Assay Kit (K-TSTA), with the blank reagent as the control, the absorbance of each sample was measured at a wavelength of 510 nm, and the amylose content was calculated. The amylose calculation formula was: amylose content (%) = ConA absorbance / total starch absorbance × 66.8.

[0111] 1.9. Wheat quality determination

[0112] 1.9.1 Protein content determination

[0113] The crude protein content of the samples was determined using a FOSS KT8000 series Kjeldahl nitrogen analyzer (FOSS, China), with 3 replicates for each material.

[0114] 1.9.2. Determination of water content

[0115] (1) The samples were measured using a HC103 rapid moisture analyzer (Mettler Toledom, Switzerland), with 3 replicates for each material.

[0116] 1.9.3. Sedimentation value determination

[0117] The samples were measured using a CAU-B tester (Zhongbangde, China), with 3 replicates for each material.

[0118] 1.9.4. RVA Viscosity Quick Analysis

[0119] The samples were measured using an RVA-Tecmaster rotary viscometer (Perth, Sweden), with 3 replicates for each material.

[0120] 1.9.5 Determination of wheat flour parameters

[0121] The farinograph 810110E series (Brabender, Germany) was used to measure the farinograph parameters. Each material was repeated three times, and 10 g of wheat flour was used for the farinograph parameter measurement each time.

[0122] 1.10. Determination of physical and chemical properties

[0123] 1.10.1 Observation of starch microstructure

[0124] The microscopic morphology of starch granules of the parent and waxy wheat was observed using a FEI Quanta 450 scanning electron microscope (FEI Company, USA).

[0125] 1.10.2 Starch granularity analysis

[0126] Starch granule size was analyzed using a laser particle size analyzer Mastersizer 3000E (Malvern, UK).

[0127] 1.10.3 Determination of the thermodynamic properties of starch

[0128] The thermodynamic properties of gelatinized starch were analyzed using a differential calorimeter DSC 2920 (TA Instruments, New Castle, DE, USA).

[0129] 1.10.4. Starch chain length distribution

[0130] Starch was analyzed using an ion chromatography system Thermo ICS5000 (Thermo Fisher Scientific, USA) with an electrochemical detector.

[0131] 1.10.5. Starch branching degree detection

[0132] The branching degree distribution of the samples was detected by 1H nuclear magnetic resonance analysis, and the instrument used was Bruker BioSpin GmbH (Bruker, USA). The branching degree was calculated using the formula "DB (%) = A / (A+B)*100", where DB is the branching degree, A is the peak area of ​​α-1,6 bonds, and B is the peak area of ​​α-1,4 bonds.

[0133] 1.10.6 Starch XRD diffraction detection

[0134] The analysis was performed using an X'Pert Pro X-ray diffractometer (PANalytical, The Netherlands).

[0135] 1.11 Data Analysis

[0136] All values ​​are expressed as mean ± standard error. IBM SPSS Statistics 21 was used to analyze the differences between samples and controls.

[0137] 2. Results and Analysis

[0138] 2.1 Rapid creation of new waxy wheat strains

[0139] 2.1.1. F1 generation obtained by hybridization of SM133 and Chuannuowai 1456

[0140] In this example, SM133 (Wx-AAbbDD) was selected as the recipient parent and Chuannuomai 1456 (Wx-aabbdd) was selected as the Wx-deficient gene donor parent to prepare a hybrid combination, and glutinous wheat with SM133 as the genetic background was created. Chuannuomai 1456 and SM133 were planted in a greenhouse, Chuannuomai 1456 was used as the male parent, and SM133 was used as the female parent for hybridization. Five plants were hybridized in the greenhouse to obtain 82 hybrids ( Figure 2 ). After obtaining the heterozygous F1 (Wx-AabbDd) generation material, subsequent generation and testing verification were carried out.

[0141] 2.1.2. Tissue culture of hybrid and backcross offspring shortens the growth period

[0142] The F1 seeds were planted in a greenhouse, and the Wx-AabbDd heterozygous plants were selected for backcrossing with SM133. 21 days after pollination of SM133, 75 seeds with milky white embryos and no damage were removed, and the seeds were sterilized and then embryos were selected ( Figure 3 After selecting the complete embryos, place them in the regeneration medium and culture them in a 25°C incubator for 15-20 days. 70 embryos emerge ( Figure 3 b), the germination rate reached 93.3%. After germination, the seedlings were transplanted to the greenhouse nutrient soil for growth, which was the BC1F1 generation hybrid material ( Figure 3 c). When transplanting BC1F1 hybrid materials, the backcross parent SM133 was planted in batches in the greenhouse. SM133 was used as the female parent and the BC1F1 generation plants were used as the male parent for backcrossing. The young embryos in the middle stage of development were taken for tissue culture regeneration and regeneration. After backcrossing twice in the same way, BC3F1 was obtained. The BC3F1 generation plants were continuously self-pollinated to the BC3F5 generation, combined with off-site summer propagation and field phenotypic identification, to screen for excellent new waxy wheat lines.

[0143] 2.1.3 Pollen iodine staining identification of plants of different generations

[0144] SM133 and Sichuan glutinous wheat 1456 are the parents, and 1 / 4 reddish-brown pollen grains are selected for backcrossing. The genotype of the SM133 parent is AAbbDD, the genotype of the all-glutinous parent Sichuan glutinous wheat 1456 is aabbdd, and the genotype of the hybrid F1 generation is AabbDd. Because the Wx-B1 of both parents is of the deletion type, only the segregation combination of the two loci Wx-A1 and Wx-D1 needs to be considered. Therefore, when 1 / 4 glutinous pollen appears in the pollen of the BC1F1 plant obtained by backcrossing F1 with SM133, it is the heterozygous type AabbDd. Pollen iodine staining was used to identify the pollen of BC1F1, BC2F1, and BC3F1 plants. Fresh pollen was iodine-stained and observed under a microscope. The reddish-brown color is glutinous wheat pollen, and the others are non-glutinous wheat pollen ( Figure 4). A heterozygous BC1F1 plant with 1 / 4 waxy pollen was selected and backcrossed with SM133 to obtain BC2F1. A heterozygous plant at the waxy site was selected and backcrossed with SM133 to obtain BC3F1. The heterozygous BC3F1 was then self-pollinated.

[0145] 2.1.4. KASP identification of homozygous offspring from self-pollination

[0146] DNA was extracted at the seedling stage of the BC3F2 generation, and the KASP molecular marker was further used to verify the Wx-1 locus of the created glutinous wheat material. According to the SNP difference sites of the parents Wx-A1 / Wx-a1 and Wx-D1 / Wx-d1, the KASP marker was developed for genotyping. The parents SM133 and Sichuan glutinous wheat 1456 were used as non-glutinous wheat and glutinous wheat controls, respectively, and ddH2O was used as a blank control for detection. The results showed that the fluorescent signal was clearly divided into three groups, among which the yellow site was a homozygous wild-type plant with the same genotype as SM133, the green site was a heterozygous plant of the Wx gene, and the blue site was a homozygous plant of glutinous wheat lacking the Wx gene, indicating that this KASP marker can accurately type the Wx-1 genotype ( Figure 5 ). 217 BC3F2 plants were typed using this marker, and 29 homozygous lines were finally screened out ( Figure 6 The above 29 lines were self-crossed to BC3F5 in the summer propagation base in Kangding City, Sichuan Province and the Wenjiang Experimental Base of Sichuan Agricultural University, and recorded as 29 waxy wheat SM133 BC3F5 lines, referred to as SM133BC3F5, and the specific numbers are shown in Table 9.

[0147] 2.1.5. Verification of homozygous offspring by iodine staining of seeds

[0148] The starch composition of the waxy wheat SM133 BC3F5 strain was further verified using the grain iodine staining method. 200 seeds of each strain were selected for grain iodine staining verification, and the phenotype was distinguished and identified based on the color after iodine staining. Non-waxy wheat was blue, and waxy wheat was reddish brown ( Figure 7). The results of grain iodine staining verification showed that all 25 strains were homozygous for glutinous properties. Due to self-pollination and generation in the experimental field, there was a certain ratio of outcrossing, and the remaining 4 strains had heterozygous grains. Among them, 6 heterozygous grains were detected in the SM133-Wx-null-112 strain, and the outcrossing ratio was 3%; 4 heterozygous grains were detected in the SM133-Wx-null-119 strain, and the outcrossing ratio was 2%; 5 heterozygous grains were detected in the SM133-Wx-null-136 strain, and the outcrossing ratio was 2.5%; 1 heterozygous grain was detected in the SM133-Wx-null-140 strain, and the outcrossing ratio was 0.5%; all the remaining grains of the 4 strains containing heterozygous grains were glutinous after iodine staining verification. Therefore, grain iodine staining can quickly identify whether there are non-glutinous heterozygous grains in the strain, thereby improving the efficiency of phenotypic identification.

[0149] 2.1.6. SDS-PAGE verification of BC3F5 homozygous strain

[0150] To ensure the accuracy of the waxy starch of the waxy wheat strains created, the Wx protein composition of each SM133 BC3F5 strain was verified again using the SDS-PAGE method. Eight SM133 BC3F5 strains (SM133-Wxn-null-54, SM133-Wxn-null-88, SM133-Wxn-null-89, SM133-Wxn-null-92, SM133-Wxn-null-127, SM133-Wxn-null-133, SM133-Wxn-null-139, SM133-Wxn-null-145) were randomly selected for protein identification. The electrophoresis pattern showed that SM133 expressed two protein bands, Wx-A1 and Wx-D1, while the Wx protein bands of the eight SM133 BC3F5 strains were completely missing. The results further proved that SM133 BC3F5 is a new waxy wheat material with complete lack of Wx protein ( Figure 8 ).

[0151] 2.2 Field performance and yield trait analysis

[0152] The agronomic traits of SM133 and SM133 BC3F5 were investigated at the heading and maturity stages. The results are shown in Tables 3-4 and Figure 9-10 The results showed that there were no significant differences between SM133 BC3F5 and SM133 in terms of plant height, flag leaf length, tiller number, and spikelet number. The spike length of SM133-Wx-null-139 was significantly higher than that of Shumai 133 (Table 3, Fig. 9 The yield traits of SM133 and SM133 BC3F5 generations were compared and analyzed in terms of grain length, grain width, surface area, and 100-grain weight (Table 4, Fig.10 ). The grain length of SM133 is 6.66mm, and the grain length of SM133 BC3F5 generation strains is between 6.23-6.48mm, among which SM133-Wx-null-133 and SM133-Wx-null-139 are shorter than the parent SM133. There is no significant difference in the grain width between SM133 and SM133 BC3F5 generation strains. The surface area of ​​SM133 BC3F5 generation strains is 17.83-18.52mm 2 Between, and the surface area of ​​SM133 19.72mm 2 There is no significant difference. The 100-grain weight of SM133 BC3F5 generation strains ranged from 4.70 to 5.01 g, which was not significantly different from that of SM133. In order to analyze the yield traits of 29 waxy wheat strains, the yields of SM133 and SM133 were measured. The yield of SM133 was 1.98 kg, and the yield of SM133 BC3F5 generation strains ranged from 1.78 to 2.42 kg. This may be due to the difference in field fertility, which led to the difference in yield from the parents (Table 4).

[0153] Table 3 Comparative analysis of agronomic traits of tested materials

[0154] Material No. Plant height (cm) Flag leaf length (cm) Tiller number Number of spikelets Ear length (cm) SM133 94.16±0.90 28.43±1.66 10±1.00 21±0.03 12.03±0.26 SM133-Wx-null-54 93.26±0.17 28.26±0.16 11±0.16 21±0.15 11.76±0.50 SM133-Wx-null-127 94.33±0.06 28.46±0.33 9±0.66 22±0.17 11.77±0.33 SM133-Wx-null-133 94.26±0.59 27.83±0.60 10±1.66 22±0.1 11.80±0.23 SM133-Wx-null-139 94.16±0.30 26.83±0.43 10±0.67 23±0.19 12.26±0.46*

[0155] Table 4 Comparative analysis of yield traits of tested materials

[0156]

[0157]

[0158] In summary, in this embodiment, the three pairs of genes Wx-A1, Wx-B1 and Wx-D1 controlling the waxy trait of wheat grains were successfully transferred to some excellent parents by the rolling backcrossing method. While obtaining the waxy genes of the donor parents, the excellent traits of the parents, such as high yield, lodging resistance, and stress resistance, were inherited, indicating that the waxy gene mutation itself does not have an adverse effect on other excellent traits such as yield.

[0159] 2.3 Analysis of the physical and chemical properties of waxy wheat

[0160] 2.3.1. Waxy wheat particle size analysis

[0161] The laser particle size analyzer was used to compare the distribution of type A starch granules (diameter > 10 μm) and type B starch granules (diameter < 10 μm) in the typical strains SM133-Wx-null-54 and SM133. Fig.11The results showed that the number of B-type starch granules in SM133-Wx-null-54 was significantly higher than that in its parent SM133, while the number of A-type starch granules was significantly lower than that in its parent SM133 ( Fig.11 ). It can be seen that the extremely significant decrease in the content of amylose causes a change in the ratio of amylose to amylopectin, which in turn affects the change in the ratio of A and B type starch granules.

[0162] 2.3.2 Analysis of starch microstructure

[0163] The microstructure of starch granules in the purified and extracted starch and grain slices of SM133-Wx-null-54 and SM133 was observed by scanning electron microscopy (SEM). Fig.12 As shown. The results showed that there were some differences in starch morphology between SM133-Wx-null-54 and SM133. Compared with SM133, the A-type and B-type starch granules in SM133-Wx-null-54 were not smooth, and the A-type and B-type were more tightly connected. By observing the starch granule morphology of SM133-Wx-null-54 and SM133 under different viewing angles, it was found that the number of A-type starch granules in SM133-Wx-null-54 was less than that in the parent SM133 ( Fig.12 ).

[0164] 2.3.3 Analysis of thermodynamic characteristics of waxy wheat strains

[0165] The thermodynamic properties of starch such as the initial gelatinization temperature (To), peak gelatinization temperature (Tp), final gelatinization temperature (Tc) and thermal enthalpy (ΔH) of SM133-Wx-nul-54 and SM133 were measured using a differential scanning calorimeter, and the results are shown in Table 5. The results show that the initial gelatinization temperature of SM133-Wx-null-54 is slightly lower than that of SM133, but the difference is not significant, but the peak gelatinization temperature (Tp), final gelatinization temperature (Tc) and thermal enthalpy (ΔH) are all significantly reduced. Amylose has a longer chain length and is difficult to gelatinize, while amylopectin has a shorter chain length and a higher degree of branching, which is easy to gelatinize. It can be seen that the amylose content in the glutinous wheat strain created by the present invention is reduced while the amylopectin content is greatly increased, so the energy required in the gelatinization process is reduced.

[0166] Table 5 Analysis of thermodynamic properties of waxy wheat

[0167]

[0168]

[0169] Note: *, * and * indicate that the differences from the parent reached 0.05 and 0.01 significant levels, respectively. To: starting temperature; Tp: peak temperature; Tc: final temperature; ΔH: thermal enthalpy.

[0170] 2.3.4 Analysis of starch chain length distribution of waxy wheat strains

[0171] In order to study the effect of the deletion of the Wx-1 gene in the waxy wheat SM133 BC3F5 generation line on the fine structure of starch, the chain length distribution of SM133-Wx-null-54 and SM133 was analyzed in this example. The results showed that the starch chain length distribution of SM133-Wx-null-54 was not significantly different from that of the parent Shumai 133 ( Fig.13 ). The branched starch molecular distribution of SM133-Wx-null-54 and SM133 was divided into four groups: DP10, DP20, DP30, and DP40 according to the distribution results. The results showed that there was no significant difference in the starch chain length distribution between the waxy wheat lines SM133-Wx-null-54 and SM133 under different branching degrees. Overall, the average polymerization degree of SM133 was 18.63±0.09 (%), and the average polymerization degree of SM133-Wx-null-54 was 18.92±0.11 (%), and there was no significant difference between the two (Table 6). The results show that the complete loss of Wx protein will not affect the distribution of starch chain length.

[0172] Table 6 Waxy wheat starch chain length distribution

[0173] Material No. DP10 DP20 DP30 DP40 Average degree of polymerization (%) SM133 0.0673 0.0369 0.0113 0.0049 18.63±0.09 SM133-Wx-null-54 0.0655 0.0362 0.0111 0.0051 18.92±0.11

[0174] Note: *, * and * indicate that the differences from the parents reach the significant level of 0.05 and 0.01 respectively.

[0175] 2.3.5 Analysis of the branching degree of waxy wheat starch

[0176] Nuclear magnetic resonance (1H) was used to analyze the starch branching degree of SM133-Wx-null-54 and SM133. Comparing the peak areas of α-1,4 and α-1,6 branched polymers, SM133 was 207723.52 and 10840.51 respectively; while SM133-Wx-null-54 was 2172.245.85 and 15185.75 respectively. The results showed that the peak area of ​​SM133-Wx-null-54 was greater than that of SM133, and it was calculated that the branching degree of SM133 was 4.96±0.14(%), while the branching degree of SM133-Wx-null-54 was 6.53±0.24(%). The branching degree of SM133-Wx-null-54 was significantly increased ( Fig.14, Table 7). Since the content of amylose in waxy wheat strains is extremely low, and the main component is amylopectin with a higher branching degree, the decrease in amylose content and the increase in amylopectin content lead to a significant increase in starch branching degree in waxy wheat strains.

[0177] Table 7 Analysis of branching degree of waxy wheat starch

[0178]

[0179]

[0180] Note: *, * and * indicate that the differences from the parents reach the significant level of 0.05 and 0.01 respectively.

[0181] 2.3.6 XRD diffraction analysis of waxy wheat starch

[0182] The crystal structure characteristics of waxy wheat and its parent SM133 were compared and analyzed using X-ray method. Characteristic diffraction peaks appeared at 15°, 17°, 18° and 23°. The diffraction peak of SM133 was the highest at 17° and then decreased, while the diffraction peak of SM133-Wx-null-54 was the highest at 15° and then decreased ( Fig.15 ), which shows that the XRD diffraction peaks of the two are stable; the crystallinity of SM133 is 16.95±0.53(%), while the crystallinity of SM133-Wx-null-54 is 18.17±0.11(%), which is higher than that of SM133, and the crystal structure is type A (Table 8). From the results, they all belong to type A crystal structure, indicating that the change of starch composition in waxy wheat lines will not change the crystal structure of starch granules.

[0183] Table 8 XRD diffraction analysis of waxy wheat starch

[0184]

[0185] Note: *, * and * indicate that the differences from the parents reach the significant level of 0.05 and 0.01 respectively.

[0186] 2.4 Quality analysis of waxy wheat

[0187] 2.4.1. Grain component analysis

[0188] The starch content and composition of 29 SM133 BC3F5 lines were measured, and the results showed that the content of amylose in SM133 was 24.22%, while the content of amylose in all SM133 BC3F5 lines was between 2.62% and 5.51%, among which the line SM133-Wxn-null-133 had the lowest content, and the content of amylose in all lines met the waxy wheat standard. The total starch content of SM133 BC3F5 lines was between 60.98% and 74.98%, and the total starch content of some SM133 BC3F5 lines was significantly higher than that of the parent SM133 (total starch content 63.32%), among which the line SM133-Wxn-null-54 was 74.98%, and the difference was very significant (Table 9). From the 29 SM133 BC3F5 lines, 4 lines with significantly increased total starch and significantly reduced amylose were selected, SM133-Wxn-null-54, SM133-Wxn-null-127, SM133-Wxn-null-133, and SM133-Wxn-null-139, and their quality indicators such as protein content, RVA viscosity analysis, and farinograph parameters were further determined. The results of protein content determination showed that the protein content of waxy wheat lines was between 11.52% and 12.22%, and the protein content of some lines was significantly higher than that of Shumai 133 (11.27%); the quality test results showed that the sedimentation value of waxy wheat lines was between 14-15mL, while that of Shumai 133 was 24mL, which was significantly lower than that of Shumai 133 (Table 10).

[0189] Table 9 Determination of starch content in test materials

[0190] serial number Material number (strain) Amylose content% Total starch content% 1 SM133-Wx-null-9 4.03±0.17** 62.57±0.68 2 SM133-Wx-null-12 4.25±0.32** 62.85±0.65 3 SM133-Wx-null-47 3.62±1.76** 61.21±0.66 4 SM133-Wx-null-54 4.62±0.71** 74.98±1.09** 5 SM133-Wx-null-73 4.92±0.01** 62.05±1.32 6 SM133-Wx-null-74 4.50±0.38** 65.59±1.44 7 SM133-Wx-null-82 5.01±0.91** 64.67±0.88 8 SM133-Wx-null-88 3.72±0.26** 66.46±0.78** 9 SM133-Wx-null-89 5.15±0.56** 67.98±0.41** 10 SM133-Wx-null-92 4.38±0.97** 67.83±0.93** 11 SM133-Wx-null-112 4.39±0.50** 70.44±0.95** 12 SM133-Wx-null-115 4.76±0.54** 66.95±1.95 13 SM133-Wx-null-127 4.01±0.15** 71.67±0.60** 14 SM133-Wx-null-129 4.11±0.09** 70.27±0.48** 15 SM133-Wx-null-131 3.44±0.36** 69.19±0.58** 16 SM133-Wx-null-133 2.62±1.50** 73.25±1.98** 17 SM133-Wx-null-136 4.44±0.92** 71.30±1.84** 18 SM133-Wx-null-139 4.36±0.74** 72.73±1.21** 19 SM133-Wx-null-140 4.60±0.06** 68.82±1.19** 20 SM133-Wx-null-145 3.87±0.38** 68.88±0.92** 21 SM133-Wx-null-151 5.51±0.28** 62.95±3.29 22 SM133-Wx-null-155 4.62±0.56** 65.81±1.17 23 SM133-Wx-null-156 5.32±0.34** 62.91±0.97 24 SM133-Wx-null-159 4.35±1.23** 60.98±1.64 25 SM133-Wx-null-210 4.52±0.85** 64.46±1.64 26 SM133-Wx-null-211 3.93±0.40** 65.01±0.78** 27 SM133-Wx-null-212 4.62±0.19** 61.20±2.47 28 SM133-Wx-null-213 4.01±0.01** 64.37±0.86 29 SM133-Wx-null-217 4.23±0.18** 60.03±0.56 Parent SM133 24.22±0.01 63.32±2.20

[0191] Note: The starch content of 9 strains of SM133 BC3F52 was compared with that of SM133. ** and ** indicate that the differences with the parent reached the significant level of 0.05 and 0.01, respectively.

[0192] Table 10 Analysis of processing quality of some new waxy wheat strains

[0193]

[0194]

[0195] Note: *, * and * indicate that the differences from the parents reach the significant level of 0.05 and 0.01 respectively.

[0196] 2.4.2 Analysis of waxy wheat RVA viscosity

[0197] The rapid viscosity analyzer (RVA) was used to measure the peak viscosity, minimum viscosity, final viscosity, rebound value, relaxation value, gelatinization temperature (℃) and peak time of four SM133BC3F5 waxy wheat lines with significantly increased total starch and significantly reduced amylose, and the results are shown in Table 11. The results showed that Shumai 133 had a peak viscosity of 3508, a minimum viscosity of 1664, a final viscosity of 2717, a relaxation value of 1844, and a peak time of 5.67; the peak viscosity of the four lines of SM133 BC3F5 was between 2195-2564, the minimum viscosity was between 576-716, the final viscosity was between 771-953, the relaxation value was between 195-249, and the peak time was between 3.47-3.53. Compared with SM133, the peak viscosity, minimum viscosity, final viscosity, relaxation value and peak time of the four waxy wheat strains were significantly reduced; and the rebound values ​​of the four strains were between 1619 and 1860, which were significantly higher than SM133 (Table 11).

[0198] Table 11 RVA characteristics of new waxy wheat lines

[0199]

[0200] Through the RVA curve ( Fig.16 ) found that: the four strains of SM133 BC3F5 generation of glutinous wheat had fast gelatinization and short peak time; the peak viscosity of the four strains of SM133 BC3F5 generation were lower than that of SM133, and the rebound value increased; after the viscosity value dropped to the trough, the viscosity curve was relatively stable, and the cooling value became smaller, which was much lower than the parent SM133. In summary, due to changes in starch composition, glutinous wheat strains exhibit starch gelatinization characteristics that are different from those of ordinary wheat. The content of amylose in glutinous wheat strains is very low, and the starch is almost entirely composed of amylopectin, which leads to significant changes in the gelatinization characteristics of starch, thereby giving glutinous wheat unique dough physical and chemical properties.

[0201] 2.4.3 Analysis of wheat flour quality parameters

[0202] The processing quality parameters of the created glutinous wheat strains were measured using a farinograph. The formation time of SM133 BC3F5 strain was 0.5-1.0 min, while that of SM133 was 1.7 min, indicating that the formation time of glutinous wheat was shorter, the gluten strength was lower, and the dough forming time was shorter; the stability time of SM133 BC3F5 strain was 0.8-1.1 min, while that of SM133 was 2.9 min, indicating that the shorter the stability time of glutinous wheat, the worse the toughness and the higher the viscosity; the water absorption rate of SM133 BC3F5 strain was 50.4-52.2%, while that of SM133 was 60.6%, indicating that the gluten strength of glutinous wheat was weak and the water absorption rate was low; SM133 The weakening degree of BC3F5 strains is 114-137FU, while that of SM133 is 91FU, indicating that the weakening degree of flour indicates that the weaker the gluten strength of glutinous wheat, the greater the weakening degree; compared with the backcross parent SM133, the farinograph parameters of formation time, stability time, water absorption rate, and weakening degree have all been greatly reduced. From the perspective of the flour quality results, some indicators of SM133 BC3F5 meet the standards of high-quality weak-gluten wheat ( Fig.17 , Table 12).

[0203] Table 12 Analysis of flour quality parameters of new waxy wheat lines

[0204] Material No. Formation time (min) Stable time (min) Water absorption (%) Consistency(FU) Weakness(FU) SM133 1.7 2.9 60.6 520 91 SM133-Wx-null-54 1.0 0.9 50.4 516 137 SM133-Wx-null-127 0.5 0.8 50.7 487 130 SM133-Wx-null-133 1.0 0.8 52.2 506 135 SM133-Wx-null-139 0.5 1.1 51.6 520 114

[0205] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A rapid breeding method for waxy wheat, characterized in that: The following steps are involved: (1) Using Shumai 133 as the female parent and low-amylose wheat as the male parent for hybridization to obtain F1 generation seeds; (2) sowing the F1 generation seeds, taking Shumai 133 as the recurrent parent, and performing backcrossing. After 15-21 days of pollination, the young embryos of the backcrossed plants were collected for tissue culture to obtain BC1F1 generation seeds; (3) sowing the BC1F1 generation seeds, identifying the pollen of the BC1F1 plants by pollen iodine staining, selecting the BC1F1 plants with reddish brown pollen, and performing backcrossing with Shumai 133 as the recurrent parent. After 15-21 days of pollination, the young embryos of the backcrossed plants were collected for tissue culture to obtain BC2F1 generation seeds; (4) Repeat step (3) once to obtain BC3F1 generation seeds; (5) sowing the BC3F1 generation seeds, identifying the pollen of the BC3F1 plants by pollen iodine staining, selecting BC3F1 plants with reddish-brown pollen for self-pollination, and taking young embryos from the self-pollinated plants for tissue culture 15-21 days after pollination to obtain BC3F2 seeds; (6) sowing the BC3F2 seeds, conducting molecular tracing of the Wx-A1 gene and the Wx-D1 gene, and then self-pollinating to obtain BC3F3 seeds homozygous for waxy wheat; (7) Repeat step (6) three times to obtain the glutinous wheat.

2. The breeding method according to claim 1, characterized in that: In steps (2) to (5), the method for preparing the culture medium used for tissue culture comprises the following steps: Mix Ms culture medium base salt, casein hydrolyzate, maltose and water in a mass volume ratio of 2.15 g:0.5 g:15 g:100 mL, and adjust the volume to 250 mL to obtain a mixture 1; 1.75 g of plant gel and water were mixed and the volume was adjusted to 250 mL to obtain mixture 2; The medium was obtained by mixing 250 mL of mixture 1, 250 mL of mixture 2, 125 μL of IBA, 62.5 μL of NAA, and 2.5 mL of vitamins.

3. The breeding method according to claim 1, characterized in that: In steps (2)-(5), the tissue culture time is 15-20 days, the temperature is 25°C, the illumination time is 16 hours, the illumination intensity is 3000-5000 Lux, and the humidity is 40%.

4. The breeding method according to claim 1, characterized in that: The molecular tracking of the Wx-A1 gene includes the following steps: using the wheat genome to be tested as a template DNA, amplifying using an upstream primer with a nucleotide sequence such as SEQ ID NO.1-2 and a downstream primer with a nucleotide sequence such as SEQ ID NO.3, and performing fluorescence scanning on the amplified product. If a HEX fluorescence signal is obtained by the fluorescence scanning, it is determined that the wheat to be tested is a wheat variety lacking the Wx-A1 gene.

5. The breeding method according to claim 1, characterized in that: The molecular tracing of the Wx-D1 gene includes the following steps: The wheat genome to be tested is used as the template DNA, and amplification is performed using upstream primers with nucleotide sequences as shown in SEQ ID NO.4-5 and downstream primers with nucleotide sequences as shown in SEQ ID NO.

6. The amplified product is subjected to fluorescence scanning. If the fluorescence scanning obtains a HEX fluorescence signal, it is determined that the wheat to be tested is a wheat variety lacking the Wx-D1 gene.

6. The breeding method according to claim 1, characterized in that: The low-amylose wheat includes Sichuan glutinous wheat 1456.

7. A primer set for identifying waxy wheat, characterized in that: The primer set includes a first primer set and a second primer set; The first primer set includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.1-2 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.3; The second primer set includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4-5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.

6.

8. Use of the primer set according to claim 7 in identifying waxy wheat varieties and / or breeding waxy wheat varieties.

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