An efficient breeding method for early-maturing highland barley

Abstract

This invention relates to the field of crop breeding technology, specifically to a highly efficient breeding method for early-maturing highland barley. The method includes the following steps: parent selection and matching, planting substrate preparation, application of growth regulators, planting management, multi-generation screening, and variety identification. This invention, by screening parents with early-maturing and disease-resistant characteristics, optimizing the planting substrate (adding microbial agents), precisely applying growth regulators, and dynamically controlling the planting environment and nutrient solution, combined with early maturity, disease resistance, and variety identification, efficiently breeds highland barley with a short growth period and strong resistance to stripe rust and powdery mildew. It optimizes plant type and yield structure, improves grain quality, achieves increased yield and genetic stability, and provides technical support for highland barley variety improvement and high-yield, high-quality cultivation.

Description

Technical Field

[0001] This invention relates to the field of crop breeding technology, specifically to an efficient breeding method for early-maturing highland barley. Background Technology

[0002] Barley, also known as naked barley because its inner and outer husks separate and the grains are exposed, is mainly distributed in high-altitude areas of my country. Its irreplaceable role in regional cultivation makes it a dominant and distinctive crop in these areas. Barley is a highly nutritious and medicinal grain, characterized by its high protein, fiber, and vitamin content, low fat, and low sugar content. It is also rich in beta-glucan, arabinoxylan, phenolic substances, phytosterols, and various beneficial minerals.

[0003] In existing technologies, barley breeding does not use planting substrates and relies mainly on conventional substrates such as natural soil or farmyard manure. The nutrient content and ratio in these substrates are random, which leads to a prolonged breeding cycle and poor trait stability, ultimately affecting breeding efficiency and quality. Traditional barley disease resistance screening relies heavily on observation of natural disease occurrence in the field, which is greatly affected by climate and pathogen prevalence, and is prone to problems such as unstable resistance identification results between years and missed screening of disease-resistant materials in years with low susceptibility. Traditional barley breeding does not actively apply growth regulators, which results in varying growth periods and excessive vegetative growth of stems and internodes, leading to taller plants but weak lodging resistance.

[0004] Based on this, the present invention provides an efficient breeding method for early-maturing highland barley. Summary of the Invention

[0005] The purpose of this invention is to provide an efficient breeding method for early-maturing highland barley. The highland barley bred by this efficient breeding method not only has a higher thousand-grain weight and effective tiller number, but also a lower stripe rust disease rate and powdery mildew incidence.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a highly efficient breeding method for early-maturing highland barley, comprising the following steps:

[0007] Parental selection and matching: Select qualified male and female parents and plant them in an appropriate ratio;

[0008] Preparation of planting substrate: The planting substrate is made of 30-50 parts of well-rotted organic fertilizer, 20-30 parts of highland barley straw powder, 10-20 parts of vermiculite and 10-20 parts of perlite. The well-rotted organic fertilizer contains 3-6% N, 2-4% P2O5, 2-4% K2O, 0.1-0.3% boron and 0.05-0.2% zinc.

[0009] Application of growth regulators: The growth regulators are gibberellins or brassinolide;

[0010] Planting Management: Implement strict management including temperature, light, water, fertilizer, and specialized nutrient solution regulation;

[0011] Multi-generation screening and variety identification: Multi-generation screening includes early maturity screening, disease resistance screening, and superior single plant screening.

[0012] Preferably, the criteria for selecting and matching the parent lines are as follows: both the male and female lines have early maturity characteristics, a growth period of ≤100 days, and the selected lines have a stripe rust disease leaf rate of ≤10% and a powdery mildew disease level of ≤1. The male line has a thousand-grain weight of ≥45g, and the female line has ≥4 effective tillers per plant. The planting ratio of the male and female lines is 1:(3-5).

[0013] Preferably, the preparation method of the planting substrate is as follows: First, the barley straw powder is crushed to a particle size of 1-3 mm. Then, the barley straw powder is mixed with well-rotted organic fertilizer, vermiculite, and perlite. 10-15% of the total mass of the substrate is added with deionized water. After stirring evenly, the mixture is placed in an autoclave and sterilized at 121°C and 0.12 MPa for 2-3 hours. After cooling to room temperature, the pH value of the substrate is tested and stabilized at 6.0-7.5. 0.5-1% of the total mass of the substrate is added with microbial inoculant, wherein the mass ratio of Bacillus subtilis to actinomycetes in the microbial inoculant is 1:1. The preparation of the planting substrate is then completed.

[0014] Preferably, the concentration of gibberellin is 50-100 mg / L, and the concentration of brassinolide is 0.01-0.1 mg / L. The application period is during the seedling stage and the early jointing stage, with the seedling stage being the 3-5 leaf stage. The application volume per hectare is 100-150 L, and the application volume per hectare at the early jointing stage is 150-200 L. The application method is uniform foliar spraying. Spraying should be done on sunny days between 9-11 am or 3-5 pm. Avoid applying nitrogen fertilizer or alkaline pesticides within 7 days before and after spraying. If it rains within 6 hours after spraying, re-spraying is required.

[0015] Preferably, the planting management parameters are as follows: sowing depth 3-5cm, row spacing 20-25cm, planting density 200,000-300,000 plants / hectare, growing period from sowing to grain filling stage, daytime temperature controlled at 18-25℃, nighttime temperature controlled at 10-15℃, daytime temperature controlled at 16-22℃ during the seedling stage, daytime temperature controlled at 21-23℃ during the jointing stage, daily light duration 10-14 hours, soil moisture content maintained at 60-80%, watering by drip irrigation, watering once every 10 days during the seedling stage, watering once every 7 days during the jointing stage, each time watering until the soil moisture content reaches 75-80%.

[0016] Preferably, during the growing season, barley-specific nutrient solution containing nitrogen, phosphorus, potassium, and trace elements is drip-irrigated every 15-20 days along with supplemental watering, at a concentration of 0.1-0.3%. The nitrogen, phosphorus, and potassium ratio of the nutrient solution is dynamically adjusted, with a ratio of 1:2:2 during the seedling stage and 3:2:3 during the jointing stage. The difference in trace element concentration between the nutrient solution and the base fertilizer is ≤0.05% and <10%. When the daytime temperature is >30℃, a shading net with a shading rate of 30-50% is used to lower the temperature. When the nighttime temperature is <8℃, the ground is covered with mulch or a thermal blanket to increase the temperature. When natural light is insufficient, LED supplemental lights are used to supplement the light to the target illumination time, with a supplemental light intensity of 30,000-50,000 lux, and the supplemental lighting time is in the morning or evening.

[0017] Preferably, the early maturity screening step in the multi-generation screening is as follows: if the parental growth period is ≤80 days, record the heading rate 45-55 days after planting; if the parental growth period is 80-100 days, record the heading rate 55-65 days after planting; select lines with a heading rate ≥90% and a time span ≤5 days; record the maturity period 80-100 days after planting; select individual plants with a maturity period 5-10 days earlier than the control variety and a maturity uniformity ≥95%; screen continuously for 3-5 generations until the difference in maturity period within the line is ≤3 days, and the coefficient of variation of the main agronomic traits of two consecutive generations of lines is ≤10%; at the same time, use 10-15 pairs of barley-specific SSR primers to detect the genetic purity of the line ≥98% to ensure genetic stability.

[0018] Preferably, the disease resistance screening steps in the multi-generation screening are as follows: during the heading stage, inoculate with a suspension of barley stripe rust spores by spraying. After inoculation, maintain a relative humidity of 85-95% and a temperature of 15-20℃. After 15-20 days, investigate the disease leaf rate and select individual plants with a disease leaf rate ≤10%. At the same time, inoculate with powdery mildew. After 20-25 days, investigate the disease severity and select individual plants with a disease severity ≤1. Conduct natural disease verification in fields severely affected by barley stripe rust and powdery mildew. Only plants with a disease leaf rate ≤10% can enter the next generation.

[0019] Preferably, the selection criteria for superior single plants in the multi-generation screening are as follows: plant height 80-100cm, stem diameter ≥5mm, and strong lodging resistance; the single plant has 3-5 effective tillers / plant, ear length 10-15cm, and ≥40 grains per ear; thousand-grain weight 40-50g, grain protein content ≥10%, and starch content ≥60%.

[0020] Preferably, the variety identification steps are as follows: determining the growth period of stable lines to confirm that the growth period is ≤100 days; conducting quality analysis to confirm that the grain protein content is ≥10%, starch content is ≥60%, and crude fiber content is ≤5%; and confirming that the resistance level to stripe rust and powdery mildew is ≤1 through disease resistance re-screening.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention uses a specially prepared planting substrate that provides a balanced and continuous supply of nitrogen, phosphorus, potassium, and trace elements through a precise ratio of well-rotted organic fertilizer and highland barley straw powder. High-pressure sterilization and microbial agents inhibit the growth of soil-borne pathogens, reducing the risk of seedling diseases and pests. At the same time, the stable pH value of the substrate creates a suitable root environment, thereby avoiding interference from environmental differences in natural soil, ensuring consistent seedling growth, and improving breeding efficiency and the stability of plant traits.

[0023] 2. This invention uses a spray method to inoculate barley stripe rust spore suspension and powdery mildew fungus, while controlling environmental conditions to avoid interference from the number of pathogens and temperature and humidity fluctuations in the natural environment on the screening results, making the disease resistance identification results of different strains more accurate. Simultaneous inoculation with stripe rust and powdery mildew allows for systematic and targeted screening of individual plants resistant to both diseases, solving the problem of inaccurate multi-disease resistance screening in traditional methods. The process of artificial inoculation + natural disease development in severely diseased fields can double verify the stability of disease resistance, preventing the next generation of strains from failing to maintain stable disease resistance in the field.

[0024] 3. In the breeding of highland barley, the application of gibberellin or brassinolide growth regulators can regulate the uniformity of the growth process, accelerate the growth and jointing; optimize plant type, inhibit excessive stem growth and promote lignification, so that the plant height and stem diameter meet the standards and enhance the lodging resistance; enhance root vitality and photosynthetic efficiency, promote the formation of effective tillers and the number of grains per ear, and work with special nutrient solution to increase the thousand-grain weight and the content of grain protein and starch. Detailed Implementation

[0025] 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.

[0026] It should be noted that all raw materials used in the following experiments are commercially available.

[0027] Example 1: A highly efficient breeding method for early-maturing highland barley, comprising the following steps:

[0028] Parental selection and matching: Select qualified male and female parents and plant them in an appropriate ratio;

[0029] Preparation of planting substrate: The planting substrate is made of 30-50 parts well-rotted organic fertilizer, 20-30 parts highland barley straw powder, 10-20 parts vermiculite and 10-20 parts perlite. The well-rotted organic fertilizer contains 3-6% N, 2-4% P2O5, 2-4% K2O, 0.1-0.3% boron and 0.05-0.2% zinc.

[0030] Application of growth regulators: The growth regulators are gibberellins or brassinolide;

[0031] Planting Management: Implement strict management including temperature, light, water, fertilizer, and specialized nutrient solution regulation;

[0032] Multi-generation screening and variety identification: Multi-generation screening includes early maturity screening, disease resistance screening, and superior single plant screening.

[0033] The criteria for selecting and matching parent lines are as follows: both the male and female lines have early maturity characteristics, a growth period of ≤100 days, and the selected lines have a stripe rust disease rate of ≤10% and a powdery mildew incidence level of ≤1. The male line has a thousand-grain weight of ≥45g, and the female line has ≥4 effective tillers per plant. The planting ratio of the male and female lines is 1:3.

[0034] The preparation method of the planting substrate is as follows: First, crush the barley straw powder to a particle size of 1mm. Then, mix the barley straw powder with well-rotted organic fertilizer, vermiculite, and perlite. Add 10% of the total substrate mass of deionized water, stir evenly, and place in an autoclave. Sterilize at 121℃ and 0.12MPa pressure for 2 hours. After cooling to room temperature, test the pH value of the substrate and stabilize it at 6.0. Add 0.5% of the total substrate mass of microbial inoculant. The mass ratio of Bacillus subtilis to actinomycetes in the microbial inoculant should be 1:1, and the total number of viable bacteria should be ≥1×10⁻⁶. 9 The bacterial agent was stirred at 300 rpm for 30 minutes to ensure uniform dispersion, thus completing the preparation of the planting substrate.

[0035] The concentration of gibberellin is 50 mg / L, and the concentration of brassinolide is 0.01 mg / L. The application period is during the seedling stage and the early jointing stage. During the seedling stage, the application volume is 100 L per hectare, and during the early jointing stage, the application volume is 150 L per hectare. The application method is to spray the leaves evenly. Spraying should be done on a sunny day at 9:00 am or 3:00 pm. Avoid applying nitrogen fertilizer or alkaline pesticides within 7 days before and after spraying. If it rains within 6 hours after spraying, re-spraying is required.

[0036] The planting and management parameters are as follows: sowing depth 3cm, row spacing 20cm, planting density 200,000 plants / hectare, growth period from sowing to grain filling, daytime temperature controlled at 18℃, nighttime temperature controlled at 10℃, daytime temperature controlled at 16℃ during the seedling stage, daytime temperature controlled at 21℃ during the jointing stage, daily light duration 10h, soil moisture content maintained at 60%, watering by drip irrigation, watering once every 10 days during the seedling stage, watering once every 7 days during the jointing stage, each time watering until the soil moisture content reaches 75%.

[0037] During the growing season, supplement water with a special nutrient solution for barley containing nitrogen, phosphorus, potassium, and trace elements every 15 days via drip irrigation. The concentration is 0.1%, and the nitrogen, phosphorus, and potassium ratio of the nutrient solution is dynamically adjusted. Specifically, the ratio is 1:2:2 during the seedling stage and 3:2:3 during the jointing stage. The difference between the concentration of trace elements and the base fertilizer should be ≤0.05% and <10%. When the daytime temperature is >30℃, use a shading net with a 30% shading rate to lower the temperature. When the nighttime temperature is <8℃, cover with mulch or insulation blankets to increase the temperature. When natural light is insufficient, use LED supplemental lighting to supplement the light to the target light duration. The supplemental lighting intensity is 30,000 lux, and the supplemental lighting time is in the morning or evening.

[0038] The steps for screening for early maturity in multi-generation screening are as follows: If the parental growth period is ≤80 days, the heading rate is recorded 45 days after planting; if the parental growth period is 80 days, the heading rate is recorded 55 days after planting. Lines with a heading rate ≥90% and a time span ≤5 days are selected. The maturity period is recorded 80 days after planting. Individual plants with a maturity period 5 days earlier than the control variety and a maturity uniformity ≥95% are selected. Screening is carried out for 3 consecutive generations until the difference in maturity period within the line is ≤3 days, and the coefficient of variation of the main agronomic traits of the line is ≤10% for two consecutive generations. At the same time, 10 pairs (number of primers) of barley-specific SSR primers are used to detect the genetic purity of the line ≥98% to ensure genetic stability.

[0039] The disease resistance screening steps in multi-generation screening are as follows: During the heading stage, inoculate using a spray method at a concentration of 1×10⁻⁶. 5 A spore suspension of *Bhutanella sporeis* (containing 100,000 spores per milliliter of suspension) was inoculated. After inoculation, the relative humidity was maintained at 85% and the temperature at 15℃. The disease incidence rate was assessed after 15 days, and individual plants with a disease incidence rate ≤10% were selected. Simultaneously, an inoculation concentration of 2×10⁻⁶ was used. 4 Powdery mildew fungus with spores / mL (containing 20,000 spores per milliliter of solution) was used. After 20 days, the disease severity was assessed. Individual plants with a disease severity of ≤1 were selected and natural disease development was verified in fields severely affected by barley stripe rust and powdery mildew. Only plants with a disease leaf rate of ≤10% were allowed to enter the next generation.

[0040] The selection criteria for superior single plants in multi-generation screening are as follows: plant height 80cm, stem diameter ≥5mm, and strong lodging resistance; effective tiller number 3 / plant, ear length 10cm, number of grains per ear ≥40; thousand-grain weight 40g, grain protein content ≥10%, and starch content ≥60%.

[0041] The variety identification steps are as follows: the growth period of stable lines is determined to be ≤100 days, quality analysis is performed, the grain protein content is ≥10%, the starch content is ≥60%, and the crude fiber content is ≤5%, and the disease resistance is screened again to confirm that the resistance level to stripe rust and powdery mildew is ≤1.

[0042] Example 2: A highly efficient breeding method for early-maturing highland barley, comprising the following steps:

[0043] Parental selection and matching: Select qualified male and female parents and plant them in an appropriate ratio;

[0044] Preparation of planting substrate: The planting substrate is made of 30-50 parts well-rotted organic fertilizer, 20-30 parts highland barley straw powder, 10-20 parts vermiculite and 10-20 parts perlite. The well-rotted organic fertilizer contains 3-6% N, 2-4% P2O5, 2-4% K2O, 0.1-0.3% boron and 0.05-0.2% zinc.

[0045] Application of growth regulators: The growth regulators are gibberellins or brassinolide;

[0046] Planting Management: Implement strict management including temperature, light, water, fertilizer, and specialized nutrient solution regulation;

[0047] Multi-generation screening and variety identification: Multi-generation screening includes early maturity screening, disease resistance screening, and superior single plant screening.

[0048] The criteria for selecting and matching parent lines are as follows: both the male and female lines have early maturity characteristics, a growth period of ≤100 days, and the selected lines have a stripe rust disease rate of ≤10% and a powdery mildew incidence level of ≤1. The male line has a thousand-grain weight of ≥45g, and the female line has ≥4 effective tillers per plant. The planting ratio of the male to female lines is 1:4.

[0049] The preparation method of the planting substrate is as follows: First, crush the barley straw powder to a particle size of 2mm. Then, mix the barley straw powder with well-rotted organic fertilizer, vermiculite, and perlite. Add 12.5% ​​of the total substrate mass of deionized water, stir evenly, and place in an autoclave. Sterilize at 121℃ and 0.12MPa pressure for 2.5 hours. After cooling to room temperature, test the pH value of the substrate and stabilize it at 6.7. Add 0.7% of the total substrate mass of microbial inoculant. The mass ratio of Bacillus subtilis to Actinomycetes in the microbial inoculant is 1:1, and the total number of viable bacteria is ≥1×10⁻⁶. 9The bacterial agent was stirred at 400 rpm for 45 minutes to ensure uniform dispersion, thus completing the preparation of the planting substrate.

[0050] The concentration of gibberellin is 75 mg / L, and the concentration of brassinolide is 0.05 mg / L. The application period is during the seedling stage and the early jointing stage. During the seedling stage, the application volume is 125 L per hectare, and during the early jointing stage, the application volume is 175 L per hectare. The application method is to spray the leaves evenly. Spraying should be done on a sunny day at 10:00 am or 4:00 pm. Avoid applying nitrogen fertilizer or alkaline pesticides within 7 days before and after spraying. If it rains within 6 hours after spraying, re-spraying is required.

[0051] The planting and management parameters are as follows: sowing depth 4cm, row spacing 22.5cm, planting density 250,000 plants / hectare, growth period from sowing to grain filling stage, daytime temperature controlled at 21℃, nighttime temperature controlled at 12℃, daytime temperature controlled at 19℃ during the seedling stage, daytime temperature controlled at 22℃ during the jointing stage, daily light duration 12h, soil moisture content maintained at 70%, watering by drip irrigation, watering once every 10 days during the seedling stage, watering once every 7 days during the jointing stage, each time watering until the soil moisture content reaches 77%.

[0052] During the growing season, supplement water with a special nutrient solution for barley containing nitrogen, phosphorus, potassium, and trace elements every 17 days via drip irrigation. The concentration is 0.2%, and the nitrogen, phosphorus, and potassium ratio of the nutrient solution is dynamically adjusted. Specifically, the ratio is 1:2:2 during the seedling stage and 3:2:3 during the jointing stage. The difference between the concentration of trace elements and the base fertilizer should be ≤0.05% and <10%. When the daytime temperature is >30℃, use a shading net with a 40% shading rate to lower the temperature. When the nighttime temperature is <8℃, cover with mulch or insulation blankets to increase the temperature. When natural light is insufficient, use LED supplemental lighting to supplement the light to the target light duration. The supplemental lighting intensity is 40,000 lux, and the supplemental lighting time is in the morning or evening.

[0053] The steps for screening for early maturity in multi-generation screening are as follows: If the parental growth period is ≤80 days, the heading rate is recorded 50 days after planting; if the parental growth period is 90 days, the heading rate is recorded 60 days after planting. Lines with a heading rate ≥90% and a time span ≤5 days are selected. The maturity period is recorded 90 days after planting. Individual plants with a maturity period 7 days earlier than the control variety and a maturity uniformity ≥95% are selected. Screening is carried out for 4 generations until the difference in maturity period within the line is ≤3 days, and the coefficient of variation of the main agronomic traits of the line is ≤10% for two consecutive generations. At the same time, 12 pairs (number of primers) of barley-specific SSR primers are used to detect the genetic purity of the line ≥98% to ensure genetic stability.

[0054] The disease resistance screening steps in multi-generation screening are as follows: During the heading stage, inoculate using a spray method at a concentration of 3×10⁻⁶. 5A spore suspension of *Bhutanella sporeis* (containing 300,000 spores per milliliter of suspension) was inoculated. After inoculation, the relative humidity was maintained at 90% and the temperature at 17℃. The disease incidence rate was assessed after 17 days, and individual plants with a disease incidence rate ≤10% were selected. Simultaneously, an inoculation concentration of 3×10⁻⁶ was used. 4 Powdery mildew fungus with spores / mL (containing 30,000 spores per milliliter of solution) was tested for disease severity after 22 days. Individual plants with disease severity ≤1 were selected for natural disease verification in fields severely affected by barley stripe rust and powdery mildew. Only plants with disease leaf rate ≤10% were allowed to enter the next generation.

[0055] The selection criteria for superior single plants in multi-generation screening are as follows: plant height 90cm, stem diameter ≥5mm, and strong lodging resistance; effective tiller number 4 / plant, ear length 12cm, number of grains per ear ≥40; thousand-grain weight 45g, grain protein content ≥10%, and starch content ≥60%.

[0056] The variety identification steps are as follows: the growth period of stable lines is determined to be ≤100 days, quality analysis is performed, the grain protein content is ≥10%, the starch content is ≥60%, and the crude fiber content is ≤5%, and the disease resistance is screened again to confirm that the resistance level to stripe rust and powdery mildew is ≤1.

[0057] Example 3: A highly efficient breeding method for early-maturing highland barley, comprising the following steps:

[0058] Parental selection and matching: Select qualified male and female parents and plant them in an appropriate ratio;

[0059] Preparation of planting substrate: The planting substrate is made of 30-50 parts well-rotted organic fertilizer, 20-30 parts highland barley straw powder, 10-20 parts vermiculite and 10-20 parts perlite. The well-rotted organic fertilizer contains 3-6% N, 2-4% P2O5, 2-4% K2O, 0.1-0.3% boron and 0.05-0.2% zinc.

[0060] Application of growth regulators: The growth regulators are gibberellins or brassinolide;

[0061] Planting Management: Implement strict management including temperature, light, water, fertilizer, and specialized nutrient solution regulation;

[0062] Multi-generation screening and variety identification: Multi-generation screening includes early maturity screening, disease resistance screening, and superior single plant screening.

[0063] The criteria for selecting and matching parent lines are as follows: both the male and female lines have early maturity characteristics, a growth period of ≤100 days, and the selected lines have a stripe rust disease rate of ≤10% and a powdery mildew incidence level of ≤1. The male line has a thousand-grain weight of ≥45g, and the female line has ≥4 effective tillers per plant. The planting ratio of the male and female lines is 1:5.

[0064] The preparation method of the planting substrate is as follows: First, crush the barley straw powder to a particle size of 3mm. Then, mix the barley straw powder with well-rotted organic fertilizer, vermiculite, and perlite. Add 15% of the total substrate mass of deionized water, stir evenly, and place in an autoclave. Sterilize at 121℃ and 0.12MPa pressure for 3 hours. After cooling to room temperature, test the pH value of the substrate and stabilize it at 7.5. Add 1% of the total substrate mass of microbial inoculant. The mass ratio of Bacillus subtilis to Actinomycetes in the microbial inoculant should be 1:1, and the total number of viable bacteria should be ≥1×10⁻⁶. 9 The bacterial agent was stirred at 500 rpm for 60 minutes to ensure uniform dispersion, thus completing the preparation of the planting substrate.

[0065] The concentration of gibberellin is 100 mg / L, and the concentration of brassinolide is 0.1 mg / L. The application period is during the seedling stage and the early jointing stage. During the seedling stage, the application volume is 150 L per hectare, and during the early jointing stage, the application volume is 200 L per hectare. The application method is to spray the leaves evenly. Spraying should be done on a sunny day at 11:00 am or 5:00 pm. Avoid applying nitrogen fertilizer or alkaline pesticides within 7 days before and after spraying. If it rains within 6 hours after spraying, re-spraying is required.

[0066] The planting and management parameters are as follows: sowing depth 5cm, row spacing 25cm, planting density 300,000 plants / hectare, growth period from sowing to grain filling, daytime temperature controlled at 25℃, nighttime temperature controlled at 15℃, daytime temperature controlled at 22℃ during the seedling stage, daytime temperature controlled at 23℃ during the jointing stage, daily light duration 14h, soil moisture content maintained at 80%, watering by drip irrigation, watering once every 10 days during the seedling stage, watering once every 7 days during the jointing stage, each time watering until the soil moisture content reaches 80%.

[0067] During the growing season, supplement water with a special nutrient solution for barley containing nitrogen, phosphorus, potassium, and trace elements every 20 days via drip irrigation. The concentration is 0.3%, and the nitrogen, phosphorus, and potassium ratio of the nutrient solution is dynamically adjusted. Specifically, the ratio is 1:2:2 during the seedling stage and 3:2:3 during the jointing stage. The difference between the concentration of trace elements and the base fertilizer should be ≤0.05% and <10%. When the daytime temperature is >30℃, use a shading net with a 50% shading rate to lower the temperature. When the nighttime temperature is <8℃, cover the ground with mulch or insulation blankets to increase the temperature. When natural light is insufficient, use LED supplemental lighting to supplement the light to the target light duration. The supplemental lighting intensity is 50,000 lux, and the supplemental lighting time is in the morning or evening.

[0068] The steps for screening for early maturity in multi-generation screening are as follows: If the parental growth period is ≤80 days, the heading rate is recorded 55 days after planting; if the parental growth period is 100 days, the heading rate is recorded 65 days after planting. Lines with a heading rate ≥90% and a time span ≤5 days are selected. The maturity period is recorded 100 days after planting. Individual plants with a maturity period 10 days earlier than the control variety and a maturity uniformity ≥95% are selected. Screening is carried out for 5 consecutive generations until the difference in maturity period within the line is ≤3 days and the coefficient of variation of the main agronomic traits of the line is ≤10% for two consecutive generations. At the same time, 15 pairs (number of primers) of barley-specific SSR primers are used to detect the genetic purity of the line ≥98% to ensure genetic stability.

[0069] The disease resistance screening steps in multi-generation screening are as follows: During the heading stage, inoculate using a spray method at a concentration of 5 × 10⁻⁶. 5 A spore suspension of *Bacillus thuringiensis* spores at a concentration of 500,000 spores per milliliter (500,000 spores per milliliter suspension) was inoculated. After inoculation, the relative humidity was maintained at 95% and the temperature at 20℃. The disease incidence rate was assessed after 20 days, and individual plants with a disease incidence rate ≤10% were selected. Simultaneously, an inoculation concentration of 5 × 10⁻⁶ spores was used. 4 Powdery mildew fungus with spores / mL (50,000 spores per milliliter of solution) was used. After 25 days, the disease severity was assessed. Individual plants with a disease severity of ≤1 were selected and natural disease development was verified in fields severely affected by barley stripe rust and powdery mildew. Only plants with a disease leaf rate of ≤10% were allowed to enter the next generation.

[0070] The selection criteria for superior single plants in multi-generation screening are as follows: plant height 100cm, stem diameter ≥5mm, and strong lodging resistance; effective tiller number 5 / plant, ear length 15cm, number of grains per ear ≥40; thousand-grain weight 50g, grain protein content ≥10%, and starch content ≥60%.

[0071] The variety identification steps are as follows: the growth period of stable lines is determined to be ≤100 days, quality analysis is performed, the grain protein content is ≥10%, the starch content is ≥60%, and the crude fiber content is ≤5%, and the disease resistance is screened again to confirm that the resistance level to stripe rust and powdery mildew is ≤1.

[0072] Comparative Example 1: The difference between this comparative example and Example 1 is that no microbial inoculant is added in this comparative example.

[0073] Comparative Example 2 differs from Example 1 in that the parent ratio in this comparative example is 1:2.

[0074] Comparative Example 3 differs from Example 1 in that no growth regulator is applied in this comparative example.

[0075] Comparative Example 4: The difference between this comparative example and Example 1 is that the nutrient solution ratio was not dynamically adjusted in this comparative example.

[0076] Barley testing: The barley selected from Examples 1, 2, 3, Comparative Examples 1, 2, 3, and 4 was tested.

[0077] 1000-grain weight test: The original sample is reduced and impurities and imperfect grains are removed to obtain a clean sample. Then, the 1000-grain method is used, that is, two groups of 1000 samples are randomly selected and placed in a container with known mass. The samples are weighed using a balance with a sensitivity of 0.01g or 0.001g. If the difference between the two groups is ≤2% of the average value, the average value is taken as the 1000-grain weight. If the difference is >2%, a second test is conducted. Finally, the average value of the group with the acceptable difference is used to calculate the 1000-grain weight. Test standard: GB / T1046.2-2008;

[0078] Effective tiller count test: Select a representative sample section or quadrat containing at least 20 plants, count the total number of effective tillers of all plants in the sample section / quadrat, and then divide by the number of plants in the sample section / quadrat to calculate the average number of effective tillers per plant. Repeat 3 times and take the average value as the result of the effective tiller count test of barley. Test basis: GB / T1633-2006.

[0079] Stripe rust disease leaf rate test: During the peak period of natural disease occurrence or the typical disease period after artificial inoculation, select representative sample plants (in the field, use diagonal or five-point sampling method, investigate at least 20 plants at each point, and a total of no less than 100 plants), check all unfolded leaves of each plant, record the number of diseased leaves with typical stripe rust symptoms and the total number of leaves investigated, calculate the disease leaf rate, repeat the investigation 3 times and take the average value, test according to: GB / T1410-2006.

[0080] Powdery mildew disease severity level test: During the peak period of disease outbreak, a five-point sampling method was used. Five sampling points were randomly selected from each plot, and 10-20 plants were investigated at each point. Disease incidence was recorded according to the 0-4 grade standard on a leaf-by-leaf basis (Grade 0: no lesions; Grade 1: lesion area ≤10% of leaf area; Grade 2: lesion area 11%-30%; Grade 3: lesion area 31%-50%; Grade 4: lesion area >50%). The number of diseased leaves at each grade was counted and the disease index was calculated. Test basis: DB54 / T0077-2014.

[0081] The obtained test data are recorded in Table 1 below:

[0082]

[0083] By comparing and analyzing the relevant data in Table 1, it can be seen that the barley bred using the efficient breeding method for early-maturing barley of this invention not only has a high thousand-grain weight and effective tiller number, reflecting high yield potential, but also low stripe rust leaf incidence and powdery mildew severity, reflecting strong resistance to these diseases. Precise parental selection lays the foundation for disease resistance and high yield, a closed-loop disease resistance system ensures stable resistance, and dynamic regulation and management synergistically improve yield. These three factors work together in terms of genetics, disease resistance, and environmental adaptation to efficiently breed early-maturing, high-quality barley. Therefore, the efficient breeding method for early-maturing barley provided by this invention has a broader market prospect and is more suitable for widespread application.

[0084] In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0085] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A highly efficient breeding method for early-maturing highland barley, characterized in that, Includes the following steps: Parental selection and ratio: Select qualified male and female parents and plant them in an appropriate ratio; the criteria for parental selection and ratio are as follows: both male and female parents have early maturity characteristics, a growth period of ≤100 days, and select strains with stripe rust disease leaf rate ≤10% and powdery mildew disease severity level ≤1, wherein the male parent has a thousand-grain weight ≥45g, and the female parent has an effective tiller number ≥4 / plant, and the planting ratio of male and female parents is 1:(3-5); Preparation of planting substrate: The planting substrate is made of 30-50 parts of well-rotted organic fertilizer, 20-30 parts of highland barley straw powder, 10-20 parts of vermiculite, and 10-20 parts of perlite. The well-rotted organic fertilizer contains 3-6% N, 2-4% P2O5, 2-4% K2O, 0.1-0.3% boron, and 0.05-0.2% zinc. The preparation method of the planting substrate is as follows: First, the highland barley straw powder is crushed to a particle size of 1-3 mm, and then the highland barley straw powder is crushed to a particle size of 1-3 mm. Straw powder is mixed with well-rotted organic fertilizer, vermiculite, and perlite. 10-15% of the total substrate mass of deionized water is added, and the mixture is stirred evenly. The mixture is then placed in an autoclave and sterilized at 121℃ and 0.12MPa pressure for 2-3 hours. After cooling to room temperature, the pH value of the substrate is tested and stabilized at 6.0-7.

5. 0.5-1% of the total substrate mass of microbial inoculant is added, wherein the mass ratio of Bacillus subtilis to actinomycetes in the microbial inoculant is 1:

1. The preparation of the planting substrate is now complete. Application of growth regulators: The growth regulators are gibberellin or brassinolide; the concentration of gibberellin is 50-100 mg / L, and the concentration of brassinolide is 0.01-0.1 mg / L. The application period is during the seedling stage and the early jointing stage, with the seedling stage being the 3-5 leaf stage. The application volume per hectare is 100-150 L, and the application volume per hectare at the early jointing stage is 150-200 L. The application method is uniform foliar spraying. Spraying should be done on sunny days between 9-11 am or 3-5 pm. Avoid applying nitrogen fertilizer or alkaline pesticides within 7 days before and after spraying. If it rains within 6 hours after spraying, re-spraying is required. Planting management: Control the sowing depth, row spacing and density, and replenish water, supplement light and apply barley nutrient solution periodically during the process; Multi-generation screening and variety identification: Multi-generation screening includes early maturity screening, disease resistance screening, and superior single plant screening. The early maturity screening steps are as follows: If the parental growth period is ≤80 days, record the heading rate 45-55 days after planting; if the parental growth period is 80-100 days, record the heading rate 55-65 days after planting. Select lines with a heading rate ≥90% and a time span ≤5 days. Record the maturity period 80-100 days after planting. Select lines that mature 5-10 days earlier than the control variety. Individual plants with a maturity uniformity of ≥95% were continuously screened for 3-5 generations until the difference in maturity within the line was ≤3 days, and the coefficient of variation of major agronomic traits between two consecutive generations was ≤10%. Simultaneously, 10-15 pairs of barley-specific SSR primers were used to test the genetic purity of the lines to ensure genetic stability (≥98%). Disease resistance screening involved spraying with a suspension of barley stripe rust spores and powdery mildew. The screening steps were as follows: during the heading stage, a suspension of barley stripe rust spores was sprayed, and after inoculation, the soil was kept clean. With a relative humidity of 85-95% and a temperature of 15-20℃, investigate the disease leaf rate after 15-20 days and select individual plants with a disease leaf rate ≤10%. Simultaneously, inoculate with powdery mildew fungus, and investigate the disease severity after 20-25 days. Select individual plants with a disease severity level ≤1 and conduct natural disease verification in fields severely affected by barley stripe rust and powdery mildew. Only plants with a disease leaf rate ≤10% can proceed to the next generation. The selection criteria for superior individual plants are: plant height 80-100cm, stem diameter ≥5mm, and strong lodging resistance. The variety has 3-5 effective tillers per plant, an ear length of 10-15cm, and ≥40 grains per ear; a thousand-grain weight of 40-50g; and grain protein content ≥10% and starch content ≥60%. The variety identification steps are as follows: determining the growth period of stable lines to confirm a growth period ≤100 days; conducting quality analysis to confirm grain protein content ≥10%, starch content ≥60%, and crude fiber content ≤5%; and confirming resistance to stripe rust and powdery mildew to ≤1 level through secondary screening.

2. The efficient breeding method for early-maturing highland barley according to claim 1, characterized in that, The planting management parameters are as follows: sowing depth 3-5cm, row spacing 20-25cm, planting density 200,000-300,000 plants / hectare, growing period from sowing to grain filling stage, daytime temperature controlled at 18-25℃, nighttime temperature controlled at 10-15℃, daytime temperature controlled at 16-22℃ during the seedling stage, daytime temperature controlled at 21-23℃ during the jointing stage, daily light duration 10-14 hours, soil moisture content maintained at 60-80%, watering by drip irrigation, watering once every 10 days during the seedling stage, watering once every 7 days during the jointing stage, each time watering until the soil moisture content reaches 75-80%.

3. The efficient breeding method for early-maturing highland barley according to claim 2, characterized in that: During the growing season, barley-specific nutrient solution containing nitrogen, phosphorus, potassium, and trace elements is drip-irrigated every 15-20 days along with supplemental watering, at a concentration of 0.1-0.3%. The nitrogen, phosphorus, and potassium ratio of the nutrient solution is dynamically adjusted, with a ratio of 1:2:2 during the seedling stage and 3:2:3 during the jointing stage. The difference in trace element concentration between the nutrient solution and the base fertilizer should be ≤0.05% and <10%. When the daytime temperature is >30℃, a shading net with a shading rate of 30-50% is used to lower the temperature. When the nighttime temperature is <8℃, the ground is covered with mulch or insulation blankets to increase the temperature. When natural light is insufficient, LED supplemental lighting is used to supplement the light to the target illumination time, with a supplemental light intensity of 30,000-50,000 lux, and the supplemental lighting time is in the morning or evening.

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