Rapid breeding method of high-dietary-fiber glutinous highland barley

Abstract

The invention relates to the technical field of crop breeding, and discloses a rapid breeding method of high-dietary-fiber glutinous highland barley, which comprises the following steps: seed pretreatment, chemical mutagenesis treatment, culture solution preparation, greenhouse culture and early comprehensive screening, rapid genetic homozygosis and high-generation accurate phenotype selection. By preparing mutagenesis liquid with specific components to induce seeds, a chemical mutagenic agent contained in the mutagenesis liquid can initiate directional variation of genetic materials of the highland barley seeds, so that the genetic basis of high dietary fibers and waxy characters is widened, and rich variant groups are provided for subsequent screening; meanwhile, in the greenhouse culture and early comprehensive screening stage, the content of beta-glucan in seeds of a single plant is subjected to nondestructive measurement by utilizing a near infrared spectrum technology, so that high-throughput and low-cost quality primary screening on a huge group in the early generation of breeding is realized, and the bottlenecks that a traditional chemical detection method is long in period, high in cost and cannot be used for early generation selection are overcome; therefore, the screening efficiency of the germplasm resources is improved.

Description

Technical Field

[0001] This invention relates to the field of crop breeding technology, specifically to a rapid breeding method for high-fiber glutinous highland barley. Background Technology

[0002] Barley is a crop unique to the Qinghai-Tibet Plateau. Thanks to its strong adaptability genes, it has not only adapted to the harsh cold and barrenness of the plateau, but also has unique nutritional value that is different from other crops. For example, the representative glutinous barley has a unique flavor and is rich in nutrients. It has the functions of lowering blood lipids, maintaining blood sugar balance, inhibiting cholesterol absorption, and reducing cardiovascular diseases.

[0003] Currently, in the breeding of highland barley varieties with high dietary fiber and glutinous quality traits, traditional field phenotypic screening of hybrid offspring and multiple generations of self-pollination with homozygotes are generally relied upon. When identifying target traits, especially dietary fiber content, in the early stages, high-throughput, non-destructive, rapid, and accurate screening cannot be achieved. Relying on later chemical testing is costly and time-consuming, which can lead to the omission of excellent intermediate materials and low breeding efficiency. At the same time, when using mutagenesis or hybridization to create variant populations, it is impossible to coordinate the simultaneous improvement and selection of the two traits of high dietary fiber and glutinousness, resulting in selected lines with single traits or poor overall traits. Furthermore, the tracking efficiency of key gene loci during the screening process is insufficient.

[0004] Therefore, a rapid breeding method for high-fiber glutinous highland barley is proposed to solve the above problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a rapid breeding method for high-fiber glutinous barley, which solves the problems mentioned in the background technology, such as the selection of strains having single or poor overall traits, and the insufficient efficiency in tracking key gene loci during the screening process.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a rapid breeding method for high-dietary-fiber glutinous highland barley, comprising the following steps:

[0007] Step 1: Seed pretreatment. Select barley seeds with a thousand-grain weight of 38g or more and a moisture content of 10%-12%. Disinfect, soak and activate the seeds.

[0008] Step 2: Chemical mutagenesis treatment. Prepare the mutagenesis solution and immerse the pretreated highland barley seeds in it for treatment. The mutagenesis time is 2-6 hours and the mutagenesis temperature is 20-30℃.

[0009] Step 3: Culture medium preparation. The culture medium is prepared to promote the growth of barley plants and the accumulation of dietary fiber.

[0010] Step 4: Greenhouse cultivation and early comprehensive screening. The mutagenized barley seeds were sown in a greenhouse culture medium, and culture solution was applied. Light, temperature and humidity conditions were controlled. In the segregating population generated by planting, superior individual plants were first screened according to agronomic traits. Then, the β-glucan content of the selected individual plant grains was non-destructively determined using near-infrared spectroscopy. Individual plants with a β-glucan content of not less than 5.0% were retained.

[0011] Step 5: Rapid genetic homozygosity. Microspore culture is performed on the single plants retained in Step 4 to obtain genetically homozygous doubled haploid lines.

[0012] Step Six: Precise Phenotypic Selection in High Generation. Among the homozygous lines obtained in Step Five, the β-glucan content and total dietary fiber content were measured, and glutinous highland barley lines with β-glucan content of not less than 6.0% and total dietary fiber content of not less than 20.0% were selected.

[0013] Preferably, the seed pretreatment in step one includes the following steps:

[0014] Soak the barley seeds in a stainless steel container containing disinfectant for 1-2 hours at room temperature. During the soaking process, stir with a magnetic stirrer at 100-200 rpm. After disinfection, remove the seeds and rinse them 3-5 times with sterile water. Then place them in an activation solution and activate them at 25-30°C for 12-24 hours.

[0015] The disinfectant solution is composed of sodium hypochlorite and distilled water, with a volume ratio of sodium hypochlorite to distilled water of 1:9 and a concentration of sodium hypochlorite of 5%-10%.

[0016] The activation solution is composed of gibberellin and distilled water, wherein the concentration of gibberellin is 50-100 mg / L and the pH value of the activation solution is 6.0-7.0.

[0017] Preferably, the chemical mutagenesis treatment in step two includes the following steps:

[0018] Select chemical mutagens such as ethyl methanesulfonate or sodium azide, and dissolve them in phosphate buffer solution with a pH of 7.0-7.5 and a molar concentration of 0.1M to prepare a mutagenic solution with a concentration of 0.1%-0.5%.

[0019] The pretreated barley seeds were completely immersed in the mutagenesis solution and placed in a shaking incubator. The seeds were shaken at 50-100 r / min, the mutagenesis temperature was controlled at 25℃±2℃, and the mutagenesis time was 2-4 hours.

[0020] After mutagenesis treatment, rinse the seeds with plenty of water 3-5 times to stop the mutagenesis reaction, and then use filter paper to absorb the surface moisture.

[0021] Preferably, in the chemical mutagenesis treatment, the preparation of the mutagenesis solution further includes adding a permeation promoter, which is dimethyl sulfoxide or polyethylene glycol, and the amount added is 1%-3% of the total volume of the mutagenesis solution. After mutagenesis, the seeds need to be restored by placing them on moist filter paper in the dark for 24 hours, and the temperature is maintained at 20-25℃.

[0022] Preferably, the culture medium in step three is made from the following raw materials in parts by weight: 40-50 parts potassium nitrate, 20-30 parts ammonium dihydrogen phosphate, 10-15 parts magnesium sulfate, 5-10 parts trace element mixture, 3-8 parts dietary fiber promoter and 2-5 parts viscosity regulator, wherein the dietary fiber promoter is pectin or cellulose derivative, and the viscosity regulator is sodium alginate or xanthan gum;

[0023] The preparation of the culture medium in step three includes the following specific steps:

[0024] Add potassium nitrate, ammonium dihydrogen phosphate and magnesium sulfate to a temperature-controlled stirring device, control the temperature at 15-25℃, stir at 300-600r / min for 15-30 minutes, then add the trace element mixture and continue stirring for 10-20 minutes.

[0025] Then add dietary fiber promoter and viscosity regulator, increase the speed to 800-1200 r / min, stir for 30-45 minutes until completely dissolved, and adjust the pH to 5.5-6.5;

[0026] The trace element mixture is composed of chelates of iron, zinc, manganese and copper, with the weight ratio of iron, zinc, manganese and copper being 1.8-2.2:0.9-1.1:0.9-1.1:0.45-0.55.

[0027] Preferably, the greenhouse cultivation in step four includes the following steps:

[0028] The culture medium is composed of vermiculite, perlite and humus in a mass ratio of 2.5-3.5:1.8-2.2:0.8-1.2, and the thickness of the medium is 10-15 cm.

[0029] The sowing density is 200-300 seeds per square meter, and the sowing depth is 2-3 cm.

[0030] During cultivation, the light intensity was set to 10,000-15,000 lux, the photocycle was 12 hours of light / 12 hours of darkness, the temperature was maintained at 25-30℃ during the day and 18-22℃ at night, and the relative humidity was controlled at 60%-80%.

[0031] The culture medium is applied every 7 days, with an application rate of 100-200 mL / m² each time. At the same time, the soil EC value and pH value are monitored regularly to ensure that the EC value is between 1.5-2.5 mS / cm and the pH value is between 6.0-7.0.

[0032] Preferably, the specific operations for screening agronomic traits in step four include:

[0033] From heading to maturity, at least three field surveys were conducted on individual plants in the segregating population.

[0034] The first survey was conducted one week after heading, mainly observing plant height, stem diameter and leaf morphology, and selecting individual plants with a height between 90-110cm, thick stems and upright leaves;

[0035] The second survey was conducted during the mid-grain filling stage, focusing on evaluating ear characteristics and stress resistance, and selecting individual plants with ear lengths greater than 10cm for two-row barley and greater than 8cm for multi-row barley, and without obvious disease patches or lodging.

[0036] The third survey was conducted at maturity, and the number of grains per ear and the thousand-grain weight were measured. Single plants with more than 26 grains per ear for two-row barley and more than 45 grains per ear for multi-row barley with a thousand-grain weight of more than 38g were selected.

[0037] Finally, based on the combined results of the three surveys, the optimal agronomic traits were selected.

[0038] Preferably, the rapid genetic homozygosity in step five includes the following steps:

[0039] Microspore culture: The anthers or free microspores of the single plants selected in step four are used as explants and inoculated into the induction medium. By inducing the production of embryoids and doubling the chromosomes, genetically homozygous doubled haploid plants are obtained. The culture temperature is 25℃±2℃, the light intensity is 2000-3000 lux, and the culture period is 30-45 days.

[0040] Preferably, step six, the high-precision phenotypic selection, includes the following steps:

[0041] Dietary fiber content detection: The total dietary fiber content in the grains was determined by enzyme gravimetric method, and the screening criterion was that the total dietary fiber content was not less than 20.0%.

[0042] Glutinousness identification: The ratio of amylose and amylopectin is determined by the iodine colorimetric method. Amylopectin ratio higher than 95% is considered glutinous.

[0043] Simultaneously, molecular marker-assisted selection was performed, using SSR or SNP markers to detect gene loci associated with high dietary fiber and glutinous texture;

[0044] Screening is completed within 20-30 days after heading, with no less than 50 grains sampled from each line.

[0045] Preferably, the enzyme gravimetric method is operated as follows:

[0046] Take 0.8-1.2g of seed sample, crush and pass through a 50-70 mesh sieve, then place it in an Erlenmeyer flask. Add phosphate buffer (pH 5.8-6.2), high-temperature amylase, protease, and amyloglucosidase sequentially for stepwise enzymatic hydrolysis. Each hydrolysis step is performed in a 36-38℃ water bath for 0.5-1.5 hours under continuous shaking. After hydrolysis, transfer the mixture to a pre-weighed sintered sand crucible for filtration. Wash the residue repeatedly with preheated distilled water (55-65℃), 90-99% ethanol, and acetone. Place the crucible and residue in a container... Dry the residue in an oven at 100-110℃ until constant weight, cool it, and weigh it. The result of this weighing is the constant weight of the residue and crucible, denoted as M1. Then, ashing the residue in a muffle furnace at 520℃ for 4 hours, cooling it, and weighing it again. The result of this weighing is the total weight of the ash and crucible after ashing, denoted as M2. Calculate the dietary fiber content by [(M1-M2-M0) / M]×100%, where M1 is the constant weight of the residue and crucible after enzymatic hydrolysis, M2 is the total weight of the ash and crucible after ashing, M0 is the constant weight of the crucible itself, and M is the weight of the grain sample.

[0047] The specific operation of the iodine colorimetric method is as follows:

[0048] Weigh 0.05-0.15g of dried and pulverized barley starch sample and place it in a test tube. Add 5-10mL of 1mol / L sodium hydroxide solution and disperse in a water bath at 85-95℃ for 10-20 minutes to completely gelatinize and dissolve the starch. After cooling, dilute to 50mL with deionized water to obtain a starch stock solution.

[0049] Pipette 1.0 mL of starch stock solution into a colorimetric tube, dilute with 25-35 mL of deionized water, then add 0.5-1.0 mL of iodine reagent, shake well immediately and avoid light exposure, and let stand at room temperature for 10-15 minutes to allow the color to develop fully.

[0050] Using a UV-Vis spectrophotometer, with the sample dilution without iodine reagent as a reference, the absorbance of the colorimetric solution was measured at wavelengths of 620 nm and 550 nm, respectively, and denoted as A. 620 and A 550 , through (A 620 -A 550 ) / (A 620 +A 550 Calculate the ratio of amylopectin to amylose.

[0051] Compared with existing technologies, this invention provides a rapid breeding method for high-fiber glutinous highland barley, which has the following beneficial effects:

[0052] 1. In this invention, during the chemical mutagenesis treatment stage, seeds are induced by preparing a mutagenic solution with specific components. The chemical mutagen contained therein can induce directional variation of the genetic material of barley seeds, broadening the genetic basis of high dietary fiber and glutinous traits, and providing a rich variant population for subsequent screening. At the same time, during the greenhouse cultivation and early comprehensive screening stages, near-infrared spectroscopy is used to non-destructively determine the β-glucan content of individual seeds, realizing high-throughput and low-cost initial quality screening of a large population in the early generation of breeding. This overcomes the bottlenecks of traditional chemical detection methods, such as long cycle, high cost, and inability to be used for early generation selection, thereby improving the screening efficiency of germplasm resources.

[0053] 2. In this invention, during the early comprehensive screening stage, multiple field surveys and comprehensive evaluations are conducted on the key agronomic traits of individual plants in the segregating population, including plant height, stem diameter, ear characteristics, and stress resistance, to ensure that the selected individual plants have excellent yield potential and field adaptability. Near-infrared spectroscopy is then used to initially screen the β-glucan content. This screening strategy, which combines "optimization of agronomic traits" with "initial screening of quality traits," achieves simultaneous and synergistic selection of multiple target traits such as high yield, stress resistance, and high quality. This avoids the limitations of single-trait selection and lays a solid foundation for cultivating breakthrough varieties with excellent comprehensive traits.

[0054] 3. In this invention, during the rapid genetic homozygosity stage, microspore culture of selected individual plants enables the rapid acquisition of doubled haploid lines with homozygous genetic backgrounds, shortening the time required for genetic homozygosity and accelerating the entire breeding process. Subsequently, in the high-generation precise phenotypic selection stage, the total dietary fiber content is determined by enzyme gravimetric method, and combined with glutinousness identification and molecular marker-assisted selection, the quality and key genotypes of homozygous lines are finally verified and selected, ensuring a high degree of consistency in dietary fiber content, glutinousness, and genetic stability of the final bred lines, thereby improving the success rate and accuracy of breeding. Detailed Implementation

[0055] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0056] Example 1: A rapid breeding method for high-fiber glutinous highland barley, comprising the following steps:

[0057] Step 1: Seed pretreatment. Select barley seeds with a thousand-grain weight of 35g and a moisture content of 10%. Disinfect, soak, and activate the seeds.

[0058] Step 2: Chemical mutagenesis treatment. Prepare the mutagenesis solution and immerse the pretreated highland barley seeds in it for treatment. The mutagenesis time is 2 hours and the mutagenesis temperature is 23℃.

[0059] Step 3: Culture medium preparation. The culture medium is prepared to promote the growth of barley plants and the accumulation of dietary fiber.

[0060] Step 4: Greenhouse cultivation and early comprehensive screening. The mutagenized barley seeds were sown in a greenhouse culture medium, and culture solution was applied. Light, temperature and humidity conditions were controlled. In the segregating population generated by planting, superior individual plants were first screened according to agronomic traits. Then, the β-glucan content of the selected individual plant grains was non-destructively determined using near-infrared spectroscopy. Individual plants with a β-glucan content of not less than 5.0% were retained.

[0061] Step 5: Rapid genetic homozygosity. Microspore culture is performed on the single plants retained in Step 4 to obtain genetically homozygous doubled haploid lines.

[0062] Step Six: Precise Phenotypic Selection in High Generation. Among the homozygous lines obtained in Step Five, the β-glucan content and total dietary fiber content were measured, and glutinous highland barley lines with β-glucan content of not less than 6.0% and total dietary fiber content of not less than 20.0% were selected.

[0063] The seed pretreatment in step one includes the following steps:

[0064] Soak the barley seeds in a stainless steel container containing disinfectant for 1 hour at room temperature. During the soaking process, stir with a magnetic stirrer at 100 r / min. After disinfection, remove the seeds, rinse them 3 times with sterile water, and then place them in an activation solution to activate at 25°C for 12 hours.

[0065] The disinfectant solution consists of sodium hypochlorite and distilled water, with a volume ratio of sodium hypochlorite to distilled water of 1:9 and a sodium hypochlorite concentration of 5%.

[0066] The activation solution consists of gibberellin and distilled water, with a gibberellin concentration of 50 mg / L and a pH of 6.0.

[0067] Step two, the chemical mutagenesis treatment, includes the following steps:

[0068] Select ethyl methanesulfonate or sodium azide as chemical mutagens and dissolve them in phosphate buffer solution with a pH of 7.0 and a molar concentration of 0.1M to prepare a 0.1% mutagenic solution. The specific preparation process is as follows: In a fume hood, slowly add the weighed chemical mutagens to the buffer solution, then place the mixture on a magnetic stirrer and stir at 300 r / min at 20°C for 30 minutes until the chemical mutagens are completely dissolved to obtain a homogeneous and clear mutagenic solution. This mutagenic solution should be prepared fresh and used within 2 hours after preparation.

[0069] The pretreated barley seeds were completely immersed in the mutagenesis solution and placed in a shaking incubator. The seeds were shaken at 50 r / min, the mutagenesis temperature was controlled at 23℃, and the mutagenesis time was 2 hours.

[0070] After the mutagenesis treatment, the seeds were rinsed three times with plenty of water to stop the mutagenesis reaction, and then the surface moisture was dried with filter paper.

[0071] In chemical mutagenesis treatment, the preparation of the mutagenesis solution also includes the addition of a permeation promoter, which is dimethyl sulfoxide or polyethylene glycol, and the amount added is 1% of the total volume of the mutagenesis solution. After mutagenesis, the seeds need to be restored by placing them on moist filter paper in the dark for 24 hours, and the temperature is maintained at 20°C.

[0072] The culture medium in step three is made from the following raw materials in parts by weight: 40 parts potassium nitrate, 20 parts ammonium dihydrogen phosphate, 10 parts magnesium sulfate, 5 parts trace element mixture, 3 parts dietary fiber promoter and 2 parts viscosity regulator. The dietary fiber promoter is pectin or cellulose derivative, and the viscosity regulator is sodium alginate or xanthan gum.

[0073] Step three, the preparation of the culture medium, includes the following specific steps:

[0074] Potassium nitrate, ammonium dihydrogen phosphate, and magnesium sulfate were added to a temperature-controlled stirring device. The temperature was controlled at 15°C, and the mixture was stirred at 300 r / min for 15 minutes. Then, the trace element mixture was added, and the stirring was continued for 10 minutes.

[0075] Then add dietary fiber promoter and viscosity regulator, increase the speed to 800 r / min, stir for 30 minutes until completely dissolved, and adjust the pH to 5.5;

[0076] The trace element mixture consists of chelates of iron, zinc, manganese, and copper, with a weight ratio of 1.8:0.9:0.9:0.45.

[0077] Step four, greenhouse cultivation, includes the following steps:

[0078] The culture medium is composed of vermiculite, perlite and humus in a mass ratio of 2.5:1.8:0.8, and the thickness of the medium is 10 cm.

[0079] The sowing density is 200 seeds per square meter, and the sowing depth is 2 cm.

[0080] During cultivation, the light intensity was set to 10,000 lux, the photocycle was 12 hours of light / 12 hours of darkness, the temperature was maintained at 25°C during the day and 18°C ​​at night, and the relative humidity was controlled at 60%.

[0081] The culture medium is applied every 7 days at a rate of 100 mL / m². At the same time, the soil EC value and pH value are monitored regularly to ensure that the EC value is 1.5 mS / cm and the pH value is 6.0.

[0082] The specific procedures for screening agronomic traits in step four include:

[0083] From heading to maturity, conduct at least three field surveys of individual plants in the segregating population;

[0084] The first survey was conducted one week after heading, mainly observing plant height, stem diameter and leaf morphology, and selecting individual plants with a height of 80cm, thick stems and upright leaves.

[0085] The second survey was conducted during the mid-grain filling stage, focusing on evaluating ear traits and stress resistance, and screening individual plants with ear lengths greater than 9 cm for two-row barley and greater than 7 cm for multi-row barley, and without obvious disease patches or lodging.

[0086] The third survey was conducted at maturity, and the number of grains per ear and the weight of a thousand grains were measured. Single plants with no less than 26 grains per ear and a weight of 40g were selected for two-row barley, and no less than 45 grains per ear and a weight of 38g or more for multi-row barley.

[0087] Finally, based on the combined results of the three surveys, the optimal agronomic traits were selected.

[0088] Step five, rapid genetic homozygosity, includes the following steps:

[0089] Microspore culture: The anthers or free microspores of the single plants selected in step four were used as explants and inoculated into the induction medium. By inducing the production of embryoids and doubling the chromosomes, genetically homozygous doubled haploid plants were obtained. The culture temperature was 23℃, the light intensity was 2000 lux, and the culture period was 30 days.

[0090] Step six, the high-precision phenotypic selection, includes the following steps:

[0091] Dietary fiber content detection: The total dietary fiber content in the grains was determined by enzyme gravimetric method, and the screening criterion was that the total dietary fiber content was not less than 20.0%.

[0092] Glutinousness identification: The ratio of amylose and amylopectin is determined by the iodine colorimetric method. Amylopectin ratio higher than 95% is considered glutinous.

[0093] Simultaneously, molecular marker-assisted selection was performed, using SSR or SNP markers to detect gene loci associated with high dietary fiber and glutinous texture;

[0094] Screening was completed within 20 days after heading, with no fewer than 50 grains sampled from each line.

[0095] The specific procedure for the enzyme gravimetric method is as follows:

[0096] Take 0.8g of seed sample, crush and pass through a 50-mesh sieve, and place it in an Erlenmeyer flask. Add phosphate buffer (pH 5.8), high-temperature amylase, protease, and amyloglucosidase sequentially for stepwise enzymatic hydrolysis. Each hydrolysis step is carried out in a 36℃ water bath for 0.5 hours under continuous shaking. After hydrolysis, transfer the mixture to a pre-weighed sintered sand crucible for vacuum filtration. Wash the residue repeatedly with preheated distilled water (55℃), 90% ethanol, and acetone. Place the crucible and residue in a 100℃ oven to dry. After reaching constant weight and cooling, weigh the residue and crucible. This weighing result is the constant weight of the residue and crucible, denoted as M1. Then, ashing the residue in a muffle furnace at 520℃ for 4 hours, cooling, and weighing again, this weighing result is the total weight of the ash and crucible after ashing, denoted as M2. The dietary fiber content is calculated by [(M1-M2-M0) / M]×100%, where M1 is the constant weight of the residue and crucible after enzymatic hydrolysis, M2 is the total weight of the ash and crucible after ashing, M0 is the constant weight of the crucible itself, and M is the weight of the grain sample.

[0097] The specific operation of the iodine colorimetric method is as follows:

[0098] Weigh 0.05g of dried and pulverized barley starch sample, place it in a test tube, add 5mL of 1mol / L sodium hydroxide solution, disperse in an 85℃ water bath for 10 minutes to completely gelatinize and dissolve the starch, cool, and then dilute to 50mL with deionized water to obtain a starch stock solution.

[0099] Pipette 1.0 mL of starch stock solution into a colorimetric tube, dilute with 25 mL of deionized water, then add 0.5 mL of iodine reagent, shake immediately and avoid light exposure, and let stand at room temperature for 10 minutes to allow the color to develop fully.

[0100] Using a UV-Vis spectrophotometer, with the sample dilution without iodine reagent as a reference, the absorbance of the colorimetric solution was measured at wavelengths of 620 nm and 550 nm, respectively, and denoted as A. 620 and A 550 , through (A 620 -A 550 ) / (A 620+A 550 Calculate the ratio of amylopectin to amylose.

[0101] Example 2: A rapid breeding method for high-fiber glutinous highland barley, comprising the following steps:

[0102] Step 1: Seed pretreatment. Select barley seeds with a thousand-grain weight of 38g and a moisture content of 11%. Disinfect, soak, and activate the seeds.

[0103] Step 2: Chemical mutagenesis treatment. Prepare the mutagenesis solution and immerse the pretreated highland barley seeds in it for treatment. The mutagenesis time is 3 hours and the mutagenesis temperature is 25℃.

[0104] Step 3: Culture medium preparation. The culture medium is prepared to promote the growth of barley plants and the accumulation of dietary fiber.

[0105] Step 4: Greenhouse cultivation and early comprehensive screening. The mutagenized barley seeds were sown in a greenhouse culture medium, and culture solution was applied. Light, temperature and humidity conditions were controlled. In the segregating population generated by planting, superior individual plants were first screened according to agronomic traits. Then, the β-glucan content of the selected individual plant grains was non-destructively determined using near-infrared spectroscopy. Individual plants with a β-glucan content of not less than 5.0% were retained.

[0106] Step 5: Rapid genetic homozygosity. Microspore culture is performed on the single plants retained in Step 4 to obtain genetically homozygous doubled haploid lines.

[0107] Step Six: Precise Phenotypic Selection in High Generation. Among the homozygous lines obtained in Step Five, the β-glucan content and total dietary fiber content were measured, and glutinous highland barley lines with β-glucan content of not less than 6.0% and total dietary fiber content of not less than 20.0% were selected.

[0108] The seed pretreatment in step one includes the following steps:

[0109] Soak the barley seeds in a stainless steel container containing disinfectant for 1.5 hours at room temperature. During the soaking process, stir with a magnetic stirrer at 150 r / min. After disinfection, remove the seeds, rinse them 4 times with sterile water, and then place them in an activation solution to activate at 27°C for 18 hours.

[0110] The disinfectant is composed of sodium hypochlorite and distilled water, with a volume ratio of sodium hypochlorite to distilled water of 1:9 and a sodium hypochlorite concentration of 7%.

[0111] The activation solution consists of gibberellin and distilled water, with a gibberellin concentration of 70 mg / L and a pH of 6.5.

[0112] Step two, the chemical mutagenesis treatment, includes the following steps:

[0113] Select ethyl methanesulfonate or sodium azide as chemical mutagens and dissolve them in phosphate buffer solution with a pH of 7.2 and a molar concentration of 0.1M to prepare a 0.3% mutagenic solution. The specific preparation process is as follows: In a fume hood, slowly add the weighed chemical mutagens to the buffer solution, then place the mixture on a magnetic stirrer and stir at 400 r / min at 23°C for 45 minutes until the chemical mutagens are completely dissolved to obtain a homogeneous and clear mutagenic solution. This mutagenic solution should be prepared fresh and used within 2 hours after preparation.

[0114] The pretreated barley seeds were completely immersed in the mutagenesis solution and placed in a shaking incubator. The seeds were shaken at 70 r / min, the mutagenesis temperature was controlled at 25℃, and the mutagenesis time was 3 hours.

[0115] After mutagenesis treatment, the seeds were rinsed four times with plenty of water to stop the mutagenesis reaction, and then the surface moisture was absorbed with filter paper.

[0116] In chemical mutagenesis treatment, the preparation of the mutagenesis solution also includes the addition of a permeation promoter, which is dimethyl sulfoxide or polyethylene glycol, at a volume of 2% of the total volume of the mutagenesis solution. After mutagenesis, the seeds need to undergo recovery culture, which is carried out in the dark on moist filter paper for 24 hours, with the temperature maintained at 23°C.

[0117] The culture medium in step three is made from the following raw materials in parts by weight: 45 parts potassium nitrate, 25 parts ammonium dihydrogen phosphate, 12 parts magnesium sulfate, 7 parts trace element mixture, 5 parts dietary fiber promoter and 3 parts viscosity regulator. The dietary fiber promoter is pectin or cellulose derivative, and the viscosity regulator is sodium alginate or xanthan gum.

[0118] Step three, the preparation of the culture medium, includes the following specific steps:

[0119] Potassium nitrate, ammonium dihydrogen phosphate, and magnesium sulfate were added to a temperature-controlled stirring device. The temperature was controlled at 20°C, and the mixture was stirred at 450 r / min for 20 minutes. Then, the trace element mixture was added, and the stirring was continued for 15 minutes.

[0120] Then add dietary fiber promoter and viscosity modifier, increase the speed to 1000r / min, stir for 40 minutes until completely dissolved, and adjust the pH to 6;

[0121] The trace element mixture consists of chelates of iron, zinc, manganese, and copper, with a weight ratio of 2:1:1:0.5.

[0122] Step four, greenhouse cultivation, includes the following steps:

[0123] The culture medium is composed of vermiculite, perlite and humus in a mass ratio of 3:2:1, and the thickness of the medium is 12cm.

[0124] The sowing density is 250 seeds per square meter, and the sowing depth is 2.5 cm.

[0125] During cultivation, the light intensity was set to 12000 lux, the photocycle was 12 hours of light / 12 hours of darkness, the temperature was maintained at 27°C during the day and 20°C at night, and the relative humidity was controlled at 70%.

[0126] The culture medium is applied every 7 days at a rate of 150 mL / m². At the same time, the soil EC value and pH value are monitored regularly to ensure that the EC value is 2 mS / cm and the pH value is 6.5.

[0127] The specific procedures for screening agronomic traits in step four include:

[0128] From heading to maturity, conduct at least three field surveys of individual plants in the segregating population;

[0129] The first survey was conducted one week after heading, mainly observing plant height, stem diameter and leaf morphology, and selecting individual plants with a height of over 90cm, thick stems and upright leaves.

[0130] The second survey was conducted during the mid-grain filling stage, focusing on evaluating ear traits and stress resistance, and selecting individual plants with ear lengths greater than 10cm for two-row barley and less than 8cm for multi-row barley, and without obvious disease patches or lodging.

[0131] The third survey was conducted at maturity, and the number of grains per ear and the weight of a thousand grains were measured. Single plants with 28 grains per ear and a weight of 42g for two-ridged barley and 48 grains per ear and a weight of 40g for multi-ridged barley were selected.

[0132] Finally, based on the combined results of the three surveys, the optimal agronomic traits were selected.

[0133] Step five, rapid genetic homozygosity, includes the following steps:

[0134] Microspore culture: The anthers or free microspores of the single plants selected in step four were used as explants and inoculated into the induction medium. By inducing the production of embryoids and doubling the chromosomes, genetically homozygous doubled haploid plants were obtained. The culture temperature was 25℃, the light intensity was 2500 lux, and the culture period was 40 days.

[0135] Step six, the high-precision phenotypic selection, includes the following steps:

[0136] Dietary fiber content detection: The total dietary fiber content in the grains was determined by enzyme gravimetric method, and the screening criterion was that the total dietary fiber content was not less than 20.0%.

[0137] Glutinousness identification: The ratio of amylose and amylopectin is determined by the iodine colorimetric method. Amylopectin ratio higher than 95% is considered glutinous.

[0138] Simultaneously, molecular marker-assisted selection was performed, using SSR or SNP markers to detect gene loci associated with high dietary fiber and glutinous texture;

[0139] Screening was completed within 25 days after heading, with no fewer than 50 grains sampled from each line.

[0140] The specific procedure for the enzyme gravimetric method is as follows:

[0141] Take 1g of seed sample, crush and pass through a 60-mesh sieve, then place it in an Erlenmeyer flask. Add phosphate buffer (pH 6), high-temperature amylase, protease, and amyloglucosidase sequentially for stepwise enzymatic hydrolysis. Each hydrolysis step is performed in a 37℃ water bath for 1 hour under continuous shaking. After hydrolysis, transfer the mixture to a pre-weighed sintered sand crucible for vacuum filtration. Wash the residue repeatedly with distilled water preheated to 60℃, 95% ethanol, and acetone. Place the crucible and residue in a 105℃ oven to dry to constant weight. After cooling, the residue and crucible were weighed. The result of this weighing was the constant weight of the residue and crucible, denoted as M1. The residue was then ashed in a muffle furnace at 520℃ for 4 hours. After cooling, it was weighed again. The result of this weighing was the total weight of the ash and crucible after ashing, denoted as M2. The dietary fiber content was calculated by [(M1-M2-M0) / M]×100%, where M1 is the constant weight of the residue and crucible after enzymatic hydrolysis, M2 is the total weight of the ash and crucible after ashing, M0 is the constant weight of the crucible itself, and M is the weight of the grain sample.

[0142] The specific operation of the iodine colorimetric method is as follows:

[0143] Weigh 0.1g of dried and pulverized barley starch sample, place it in a test tube, add 7mL of 1mol / L sodium hydroxide solution, disperse in a 90℃ water bath for 15 minutes to completely gelatinize and dissolve the starch, cool, and then dilute to 50mL with deionized water to obtain starch stock solution.

[0144] Pipette 1.0 mL of starch stock solution into a colorimetric tube, dilute with 30 mL of deionized water, then add 0.7 mL of iodine reagent, shake immediately and avoid light exposure, and let stand at room temperature for 12 minutes to allow the color to develop fully.

[0145] Using a UV-Vis spectrophotometer, with the sample dilution without iodine reagent as a reference, the absorbance of the colorimetric solution was measured at wavelengths of 620 nm and 550 nm, respectively, and denoted as A. 620 and A 550 , through (A 620 -A 550 ) / (A620 +A 550 Calculate the ratio of amylopectin to amylose.

[0146] Example 3: A rapid breeding method for high-fiber glutinous highland barley, comprising the following steps:

[0147] Step 1: Seed pretreatment. Select barley seeds with a thousand-grain weight of 45g and a moisture content of 12%. Disinfect, soak, and activate the seeds.

[0148] Step 2: Chemical mutagenesis treatment. Prepare the mutagenesis solution and immerse the pretreated highland barley seeds in it for treatment. The mutagenesis time is 4 hours and the mutagenesis temperature is 27℃.

[0149] Step 3: Culture medium preparation. The culture medium is prepared to promote the growth of barley plants and the accumulation of dietary fiber.

[0150] Step 4: Greenhouse cultivation and early comprehensive screening. The mutagenized barley seeds were sown in a greenhouse culture medium, and culture solution was applied. Light, temperature and humidity conditions were controlled. In the segregating population generated by planting, superior individual plants were first screened according to agronomic traits. Then, the β-glucan content of the selected individual plant grains was non-destructively determined using near-infrared spectroscopy. Individual plants with a β-glucan content of not less than 5.0% were retained.

[0151] Step 5: Rapid genetic homozygosity. Microspore culture is performed on the single plants retained in Step 4 to obtain genetically homozygous doubled haploid lines.

[0152] Step Six: Precise Phenotypic Selection in High Generation. Among the homozygous lines obtained in Step Five, the β-glucan content and total dietary fiber content were measured, and glutinous highland barley lines with β-glucan content of not less than 6.0% and total dietary fiber content of not less than 20.0% were selected.

[0153] The seed pretreatment in step one includes the following steps:

[0154] Soak the barley seeds in a stainless steel container containing disinfectant for 2 hours at room temperature. During the soaking process, stir with a magnetic stirrer at 200 r / min. After disinfection, remove the seeds, rinse them 5 times with sterile water, and then place them in an activation solution to activate at 30°C for 24 hours.

[0155] The disinfectant solution consists of sodium hypochlorite and distilled water, with a volume ratio of sodium hypochlorite to distilled water of 1:9 and a sodium hypochlorite concentration of 10%.

[0156] The activation solution consists of gibberellin and distilled water, with a gibberellin concentration of 100 mg / L and a pH of 7.0.

[0157] Step two, the chemical mutagenesis treatment, includes the following steps:

[0158] Select ethyl methanesulfonate or sodium azide as chemical mutagens and dissolve them in phosphate buffer solution with a pH of 7.5 and a molar concentration of 0.1M to prepare a 0.5% mutagenic solution. The specific preparation process is as follows: In a fume hood, slowly add the weighed chemical mutagens to the buffer solution, then place the mixture on a magnetic stirrer and stir at 500 r / min for 60 minutes at 25°C until the chemical mutagens are completely dissolved to obtain a homogeneous and clear mutagenic solution. This mutagenic solution should be prepared fresh and used within 2 hours after preparation.

[0159] The pretreated barley seeds were completely immersed in the mutagenesis solution and placed in a shaking incubator. The seeds were shaken at 100 r / min, the mutagenesis temperature was controlled at 27℃, and the mutagenesis time was 4 hours.

[0160] After the mutagenesis treatment, the seeds were rinsed five times with plenty of water to stop the mutagenesis reaction, and then the surface moisture was absorbed with filter paper.

[0161] In chemical mutagenesis treatment, the preparation of the mutagenesis solution also includes the addition of a permeation promoter, which is dimethyl sulfoxide or polyethylene glycol, and the amount added is 3% of the total volume of the mutagenesis solution. After mutagenesis, the seeds need to be restored by placing them on moist filter paper in the dark for 24 hours, and the temperature is maintained at 25°C.

[0162] The culture medium in step three is made from the following raw materials in parts by weight: 50 parts potassium nitrate, 30 parts ammonium dihydrogen phosphate, 15 parts magnesium sulfate, 10 parts trace element mixture, 8 parts dietary fiber promoter and 5 parts viscosity regulator. The dietary fiber promoter is pectin or cellulose derivative, and the viscosity regulator is sodium alginate or xanthan gum.

[0163] Step three, the preparation of the culture medium, includes the following specific steps:

[0164] Potassium nitrate, ammonium dihydrogen phosphate, and magnesium sulfate were added to a temperature-controlled stirring device. The temperature was controlled at 25°C, and the mixture was stirred at 600 r / min for 30 minutes. Then, the trace element mixture was added, and the stirring was continued for another 20 minutes.

[0165] Then add dietary fiber promoter and viscosity regulator, increase the speed to 1200 r / min, stir for 45 minutes until completely dissolved, and adjust the pH to 6.5;

[0166] The trace element mixture consists of chelates of iron, zinc, manganese, and copper, with a weight ratio of 2.2:1.1:1.1:0.55.

[0167] Step four, greenhouse cultivation, includes the following steps:

[0168] The culture medium is composed of vermiculite, perlite and humus in a mass ratio of 3.5:2.2:1.2, and the substrate thickness is 15cm.

[0169] The sowing density is 300 seeds per square meter, and the sowing depth is 3 cm.

[0170] During cultivation, the light intensity was set to 15,000 lux, the photocycle was 12 hours of light / 12 hours of darkness, the temperature was maintained at 30°C during the day and 22°C at night, and the relative humidity was controlled at 80%.

[0171] The culture medium was applied every 7 days at a rate of 200 mL / m². Soil EC and pH values ​​were monitored regularly to ensure that the EC value was 2.5 mS / cm and the pH value was 7.0.

[0172] The specific procedures for screening agronomic traits in step four include:

[0173] From heading to maturity, conduct at least three field surveys of individual plants in the segregating population;

[0174] The first survey was conducted one week after heading, mainly observing plant height, stem diameter and leaf morphology, and selecting individual plants with a height of 95cm, thick stems and upright leaves.

[0175] The second survey was conducted during the mid-grain filling stage, focusing on evaluating ear traits and stress resistance, and selecting individual plants with a two-row barley ear length of 12cm and a multi-row barley ear length of 10cm, and without obvious disease patches or lodging.

[0176] The third survey was conducted at maturity. The number of grains per ear and the weight of a thousand grains were measured. Individual plants with 30 grains per ear and a weight of 45g were selected for two-ridged barley, and individual plants with 50 grains per ear and a weight of 42g were selected for multi-ridged barley.

[0177] Finally, based on the combined results of the three surveys, the optimal agronomic traits were selected.

[0178] Step five, rapid genetic homozygosity, includes the following steps:

[0179] Microspore culture: The anthers or free microspores of the single plants selected in step four were used as explants and inoculated into the induction medium. By inducing the production of embryoids and doubling the chromosomes, genetically homozygous doubled haploid plants were obtained. The culture temperature was 27℃, the light intensity was 3000 lux, and the culture period was 45 days.

[0180] Step six, the high-precision phenotypic selection, includes the following steps:

[0181] Dietary fiber content detection: The total dietary fiber content in the grains was determined by enzyme gravimetric method, and the screening criterion was that the total dietary fiber content was not less than 20.0%.

[0182] Glutinousness identification: The ratio of amylose and amylopectin is determined by the iodine colorimetric method. Amylopectin ratio higher than 95% is considered glutinous.

[0183] Simultaneously, molecular marker-assisted selection was performed, using SSR or SNP markers to detect gene loci associated with high dietary fiber and glutinous texture;

[0184] Screening was completed within 30 days after heading, with no fewer than 50 grains sampled from each line.

[0185] The specific procedure for the enzyme gravimetric method is as follows:

[0186] Take 1.2g of seed sample, crush and pass through a 70-mesh sieve, and place it in an Erlenmeyer flask. Add phosphate buffer (pH 6.2), high-temperature amylase, protease, and amyloglucosidase sequentially for stepwise enzymatic hydrolysis. Each hydrolysis step is carried out in a 38℃ water bath for 1.5 hours under continuous shaking. After hydrolysis, transfer the mixture to a pre-weighed sintered sand crucible for vacuum filtration. Wash the residue repeatedly with distilled water preheated to 65℃, 99% ethanol, and acetone. Place the crucible and residue in a 110℃ oven to dry. After reaching constant weight and cooling, weigh the residue and crucible. This weighing result is the constant weight of the residue and crucible, denoted as M1. Then, ashing the residue in a muffle furnace at 520℃ for 4 hours, cooling, and weighing again, this weighing result is the total weight of the ash and crucible after ashing, denoted as M2. The dietary fiber content is calculated by [(M1-M2-M0) / M]×100%, where M1 is the constant weight of the residue and crucible after enzymatic hydrolysis, M2 is the total weight of the ash and crucible after ashing, M0 is the constant weight of the crucible itself, and M is the weight of the grain sample.

[0187] The specific operation of the iodine colorimetric method is as follows:

[0188] Weigh 0.15g of dried and pulverized barley starch sample, place it in a test tube, add 10mL of 1mol / L sodium hydroxide solution, disperse in a 95℃ water bath for 20 minutes to completely gelatinize and dissolve the starch, cool, and then dilute to 50mL with deionized water to obtain starch stock solution.

[0189] Pipette 1.0 mL of starch stock solution into a colorimetric tube, dilute with 35 mL of deionized water, then add 1.0 mL of iodine reagent, shake immediately and avoid light exposure, and let stand at room temperature for 15 minutes to allow the color to develop fully.

[0190] Using a UV-Vis spectrophotometer, with the sample dilution without iodine reagent as a reference, the absorbance of the colorimetric solution was measured at wavelengths of 620 nm and 550 nm, respectively, and denoted as A. 620 and A 550 , through (A 620 -A 550 ) / (A620 +A 550 Calculate the ratio of amylopectin to amylose.

[0191] Comparative Example 1: The difference between this comparative example and Example 1 is that no mutagenic solution was used in the chemical mutagenesis treatment of this comparative example.

[0192] Comparative Example 2 differs from Example 1 in that no dietary fiber promoter was added when preparing the culture medium in this comparative example.

[0193] Comparative Example 3 differs from Example 1 in that the selected individual plants were not cultured into microspores in this comparative example.

[0194] Comparative Example 4 differs from Example 1 in that near-infrared spectroscopy was not used to determine the amount of β-glucan 542B during the early comprehensive screening of this comparative example.

[0195] The breeding efficacy and quality of the high-dietary-fiber glutinous highland barley stable lines bred in Examples 1-3 and Comparative Examples 1-4 were tested. The test items and methods are as follows:

[0196] The phenotypic variation rate test was conducted by observing and measuring four key agronomic traits in the M1 generation population after mutagenesis treatment: plant height, number of effective tillers, ear length, and heading date. The mean, standard deviation, and coefficient of variation of each trait in the population were calculated and compared with the corresponding data of the original parent population without mutagenesis to assess the breadth of phenotypic variation induced by the mutagenesis treatment.

[0197] To test the activity of key enzymes in dietary fiber synthesis, flag leaf and grain samples were collected from highland barley during the mid-grain-filling stage. The key active units of cellulose synthase and β-glucan synthase were determined by enzyme-linked immunosorbent assay (ELISA) or spectrophotometry, and the differences in enzyme activity among different treatment groups were compared.

[0198] Genetic homozygosity and trait stability testing: During the strain selection stage, whole-genome SNP markers are used to scan the strains and calculate their genetic homozygosity percentage.

[0199] To test the efficiency and accuracy of early-generation screening, two methods were used in the F2 segregating population: rapid screening using near-infrared spectroscopy and traditional chemical detection. The β-glucan content of the same batch of individual plants was measured, and the time, economic cost, and correlation of the results of the two methods were compared. Finally, the results of chemical detection in later generations were used as the gold standard to calculate the accuracy and false negative rate of early near-infrared screening.

[0200] The test data of the high dietary fiber glutinous highland barley selected in Examples 1-3 and Comparative Examples 1-4 are recorded in the table below:

[0201] Table 1: Test Data Record Table

[0202]

[0203] Comparison and analysis of the data in the table show that the high-fiber glutinous highland barley lines bred using the methods in Examples 1-3 outperformed the results obtained in Comparative Examples 1-4 in terms of phenotypic variation rate, key enzyme activity, genetic homozygosity, and screening accuracy. This indicates that in the chemical mutagenesis stage, the seeds were induced by preparing a mutagenic solution with specific components. The chemical mutagen contained in the solution could induce directional variation in the genetic material of highland barley seeds, broadening the genetic basis for high dietary fiber and glutinous traits, and providing a rich variant population for subsequent screening. At the same time, in the greenhouse cultivation and early comprehensive screening stages, the β-glucan content of individual seeds was non-destructively measured using near-infrared spectroscopy, enabling high-throughput and low-cost initial quality screening of a large population in the early generations of breeding. This overcame the bottlenecks of traditional chemical detection methods, such as long cycles, high costs, and inability to be used for early generation selection, thereby improving the screening efficiency of germplasm resources. In the early comprehensive screening stage, multiple field surveys and comprehensive evaluations were conducted on key agronomic traits of individual plants in the segregating population, including plant height, stem diameter, ear characteristics, and stress resistance, to ensure that the selected individual plants had excellent yield potential and field adaptability. Near-infrared spectroscopy was then used for preliminary screening of β-glucan content. This screening strategy, combining "optimization of agronomic traits" with "preliminary screening of quality traits," achieved simultaneous and synergistic selection of multiple target traits such as high yield, stress resistance, and high quality, avoiding the limitations of single-trait selection and laying a solid foundation for cultivating breakthrough varieties with excellent comprehensive traits. In the rapid genetic homozygosity stage, microspore culture of selected individual plants can quickly yield doubled haploid lines with homozygous genetic backgrounds, shortening the time required for genetic homozygosity and accelerating the entire breeding process. Subsequently, in the high-generation precise phenotypic selection stage, the total dietary fiber content is determined by enzyme gravimetric method, and combined with glutinousness identification and molecular marker-assisted selection, the quality and key genotypes of homozygous lines are finally verified and selected, ensuring a high degree of consistency in dietary fiber content, glutinousness, and genetic stability of the final bred lines, thus improving the success rate and accuracy of breeding.

[0204] By comparing and analyzing the relevant data in the table, it can be seen that the high-dietary-fiber glutinous barley strain obtained by the breeding method of this invention exhibits advantages in multiple dimensions, including genetic gain, trait stability, and breeding efficiency. This demonstrates that the rapid breeding method provided by this invention has higher efficiency and reliability.

[0205] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0206] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A rapid breeding method for high-dietary-fiber glutinous highland barley, characterized in that: Includes the following steps: Step 1: Seed pretreatment. Select barley seeds with a thousand-grain weight of 35-45g and a moisture content of 10%-12%. Disinfect, soak, and activate the seeds. Step 2: Chemical mutagenesis treatment. Prepare the mutagenesis solution and immerse the pretreated highland barley seeds in it for treatment. The mutagenesis time is 2-6 hours and the mutagenesis temperature is 20-30℃. Step 3: Culture medium preparation. The culture medium is prepared to promote the growth of barley plants and the accumulation of dietary fiber. Step 4: Greenhouse cultivation and early comprehensive screening. The mutagenized barley seeds were sown in a greenhouse culture medium, and culture solution was applied. Light, temperature and humidity conditions were controlled. In the segregating population generated by planting, superior individual plants were first screened according to agronomic traits. Then, the β-glucan content of the selected individual plant grains was non-destructively determined using near-infrared spectroscopy. Individual plants with a β-glucan content of not less than 5.0% were retained. Step 5: Rapid genetic homozygosity. Microspore culture is performed on the single plants retained in Step 4 to obtain genetically homozygous doubled haploid lines. Step Six: Precise Phenotypic Selection in High Generation. Among the homozygous lines obtained in Step Five, the β-glucan content and total dietary fiber content were measured, and glutinous highland barley lines with β-glucan content of not less than 6.0% and total dietary fiber content of not less than 20.0% were selected.

2. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: The seed pretreatment in step one includes the following steps: Soak the barley seeds in a stainless steel container containing disinfectant for 1-2 hours at room temperature. During the soaking process, stir with a magnetic stirrer at 100-200 rpm. After disinfection, remove the seeds and rinse them 3-5 times with sterile water. Then place them in an activation solution and activate them at 25-30°C for 12-24 hours. The disinfectant solution is composed of sodium hypochlorite and distilled water, with a volume ratio of sodium hypochlorite to distilled water of 1:9 and a concentration of sodium hypochlorite of 5%-10%. The activation solution is composed of gibberellin and distilled water, wherein the concentration of gibberellin is 50-100 mg / L and the pH value of the activation solution is 6.0-7.

0.

3. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: The chemical mutagenesis treatment in step two includes the following steps: Select chemical mutagens such as ethyl methanesulfonate or sodium azide, and dissolve them in phosphate buffer solution with a pH of 7.0-7.5 and a molar concentration of 0.1M to prepare a mutagenic solution with a concentration of 0.1%-0.5%. The pretreated barley seeds were completely immersed in the mutagenesis solution and placed in a shaking incubator. The seeds were shaken at 50-100 r / min, the mutagenesis temperature was controlled at 25℃±2℃, and the mutagenesis time was 2-4 hours. After mutagenesis treatment, rinse the seeds with plenty of water 3-5 times to stop the mutagenesis reaction, and then use filter paper to absorb the surface moisture.

4. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 3, characterized in that: In the chemical mutagenesis treatment, the preparation of the mutagenesis solution also includes the addition of a permeation promoter, which is dimethyl sulfoxide or polyethylene glycol, and the amount added is 1%-3% of the total volume of the mutagenesis solution. After mutagenesis, the seeds need to be restored by placing them on moist filter paper in the dark for 24 hours, and the temperature is maintained at 20-25℃.

5. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: The culture medium in step three is made from the following raw materials in parts by weight: 40-50 parts potassium nitrate, 20-30 parts ammonium dihydrogen phosphate, 10-15 parts magnesium sulfate, 5-10 parts trace element mixture, 3-8 parts dietary fiber promoter and 2-5 parts viscosity regulator. The dietary fiber promoter is pectin or cellulose derivative, and the viscosity regulator is sodium alginate or xanthan gum. The preparation of the culture medium in step three includes the following specific steps: Add potassium nitrate, ammonium dihydrogen phosphate and magnesium sulfate to a temperature-controlled stirring device, control the temperature at 15-25℃, stir at 300-600r / min for 15-30 minutes, then add the trace element mixture and continue stirring for 10-20 minutes. Then add dietary fiber promoter and viscosity regulator, increase the speed to 800-1200 r / min, stir for 30-45 minutes until completely dissolved, and adjust the pH to 5.5-6.5; The trace element mixture is composed of chelates of iron, zinc, manganese and copper, with the weight ratio of iron, zinc, manganese and copper being 1.8-2.2:0.9-1.1:0.9-1.1:0.45-0.

55.

6. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: Greenhouse cultivation in step four Includes the following steps: The culture medium is composed of vermiculite, perlite and humus in a mass ratio of 2.5-3.5:1.8-2.2:0.8-1.2, and the thickness of the medium is 10-15 cm. The sowing density is 200-300 seeds per square meter, and the sowing depth is 2-3 cm. During cultivation, the light intensity was set to 10,000-15,000 lux, the photocycle was 12 hours of light / 12 hours of darkness, the temperature was maintained at 25-30℃ during the day and 18-22℃ at night, and the relative humidity was controlled at 60%-80%. The culture medium is applied every 7 days, with an application rate of 100-200 mL / m² each time. At the same time, the soil EC value and pH value are monitored regularly to ensure that the EC value is between 1.5-2.5 mS / cm and the pH value is between 6.0-7.

0.

7. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: The specific operations for screening agronomic traits in step four include: From heading to maturity, at least three field surveys were conducted on individual plants in the segregating population. The first survey was conducted one week after heading, mainly observing plant height, stem diameter and leaf morphology, and selecting individual plants with a height between 90-110cm, thick stems and upright leaves; The second survey was conducted during the mid-grain filling stage, focusing on evaluating ear characteristics and stress resistance. Individual plants with ear lengths greater than 10 cm for two-row barley and greater than 8 cm for multi-row barley were selected, and those without obvious disease patches or lodging were selected. The third survey was conducted at maturity, and the number of grains per ear and the weight of a thousand grains were measured. Single plants with more than 26 grains per ear for two-row barley and more than 45 grains per ear for multi-row barley with a weight of not less than 38g per thousand grains were selected. Finally, based on the combined results of the three surveys, the optimal agronomic traits were selected.

8. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: Step five, rapid genetic homozygosity, includes the following steps: Microspore culture: The anthers or free microspores of the single plants selected in step four are used as explants and inoculated into the induction medium. By inducing the production of embryoids and doubling the chromosomes, genetically homozygous doubled haploid plants are obtained. The culture temperature is 25℃±2℃, the light intensity is 2000-3000 lux, and the culture period is 30-45 days.

9. The rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 1, characterized in that: Step six, the high-precision phenotypic selection, includes the following steps: Dietary fiber content detection: The total dietary fiber content in the grains was determined by enzyme gravimetric method, and the screening criterion was that the total dietary fiber content was not less than 20.0%. Glutinousness identification: The ratio of amylose and amylopectin is determined by the iodine colorimetric method. Amylopectin ratio higher than 95% is considered glutinous. Simultaneously, molecular marker-assisted selection was performed, using SSR or SNP markers to detect gene loci associated with high dietary fiber and glutinous texture; Screening is completed within 20-30 days after heading, with no less than 50 grains sampled from each line.

10. A rapid breeding method for high-dietary-fiber glutinous highland barley according to claim 9, characterized in that: The specific operation of the enzyme gravimetric method is as follows: Take 0.8-1.2g of seed sample, crush and pass through a 50-70 mesh sieve, then place it in an Erlenmeyer flask. Add phosphate buffer (pH 5.8-6.2), high-temperature amylase, protease, and amyloglucosidase sequentially for stepwise enzymatic hydrolysis. Each hydrolysis step is performed in a 36-38℃ water bath for 0.5-1.5 hours under continuous shaking. After hydrolysis, transfer the mixture to a pre-weighed sintered sand crucible for filtration. Wash the residue repeatedly with preheated distilled water (55-65℃), 90-99% ethanol, and acetone. Place the crucible and residue in a container... Dry the residue in an oven at 100-110℃ until constant weight, cool it, and weigh it. The result of this weighing is the constant weight of the residue and crucible, denoted as M1. Then, ashing the residue in a muffle furnace at 520℃ for 4 hours, cooling it, and weighing it again. The result of this weighing is the total weight of the ash and crucible after ashing, denoted as M2. Calculate the dietary fiber content by [(M1-M2-M0) / M]×100%, where M1 is the constant weight of the residue and crucible after enzymatic hydrolysis, M2 is the total weight of the ash and crucible after ashing, M0 is the constant weight of the crucible itself, and M is the weight of the grain sample. The specific operation of the iodine colorimetric method is as follows: Weigh 0.05-0.15g of dried and pulverized barley starch sample, place it in a test tube, add 5-10mL of 1mol / L sodium hydroxide solution, and disperse it in a water bath at 85-95℃ for 10-20 minutes to completely gelatinize and dissolve the starch. After cooling, dilute to 50mL with deionized water to obtain a starch stock solution. Pipette 1.0 mL of starch stock solution into a colorimetric tube, dilute with 25-35 mL of deionized water, then add 0.5-1.0 mL of iodine reagent, shake well immediately and avoid light exposure, and let stand at room temperature for 10-15 minutes to allow the color to develop fully. Using a UV-Vis spectrophotometer, with the sample dilution without iodine reagent as a reference, the absorbance of the colorimetric solution was measured at wavelengths of 620 nm and 550 nm, respectively, and denoted as A. 620 and A 550 , through (A 620 -A 550 ) / (A 620 +A 550 Calculate the ratio of amylopectin to amylose.

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