Breeding method of sorghum variety for brewing wine

Abstract

The invention relates to the technical field of sorghum variety breeding, and discloses a sorghum variety breeding method for wine brewing, which comprises the following steps: determining quality, yield, resistance and adaptability targets, collecting and innovating sorghum germplasm through traditional and modern breeding technologies according to the targets, and producing new strain materials matched with the targets; performing agronomic character and basic physicochemical index evaluation on the new strain material, screening out standard strains, and performing metabonomics analysis and differential metabolite screening through UPLC-MS / MS to obtain candidate strains; carrying out regional and production tests on the screened candidate strains in multiple ecological regions to verify the yielding ability, the yield stability and the stress resistance of the candidate strains, and then carrying out accurate quality reinspection and Maotai-flavor liquor small-scale test brewing on the ecological adaptive strains to obtain the high-quality sorghum strain for special liquor. The method effectively solves the problems of aggravated plant diseases and insect pests, hybrid and degeneration of varieties, reduction of high yield performance and deterioration of quality caused by long-term single planting of mainstream varieties and lack of systematic purification and rejuvenation in the current production of the sorghum for wine.

Description

Technical Field

[0001] This invention relates to the field of sorghum variety breeding technology, and in particular to a method for breeding sorghum varieties for brewing. Background Technology

[0002] Currently, in the production of sorghum for liquor, a certain mainstream variety has suffered from problems due to long-term monoculture and large-scale planting, coupled with a lack of systematic purification and rejuvenation technologies. These problems include increasingly severe pest and disease outbreaks, reduced seed purity, varietal contamination and degeneration, decreased yield, and continuous quality deterioration. These issues further trigger a chain reaction, leading to the need for additional pesticides to control pests and diseases, multiple harvests due to inconsistent varietal growth and maturity, and reduced alcohol yield and quality during brewing. Ultimately, this results in a predicament of low yield, poor quality, weak resistance, high production costs, and low farmer income, failing to meet the demand of brewing enterprises for stable yields and high-quality sorghum for liquor production. This severely restricts the improvement of agricultural production efficiency and the sustainable development of the liquor industry.

[0003] In terms of variety selection, existing sorghum breeding technology for liquor suffers from several shortcomings, including insufficient multidisciplinary collaboration, low compatibility between breeding methods and brewing process requirements, and a lack of techniques for preventing contamination and maintaining purity in the propagation of superior varieties. On the one hand, the integration of traditional breeding with modern biotechnology is not high, and there is a lack of systematic collaboration among research forces from multiple disciplines such as breeding, cultivation, soil and fertilizer, physiology, nutrition, and plant diseases, making it difficult to efficiently and directionally select varieties with excellent traits such as short stalks, large ears, early maturity, and disease resistance. On the other hand, the existing breeding process does not pay enough attention to the compatibility with the specific brewing process of Maotai-flavor liquor, and lacks targeted quality testing and brewing verification. At the same time, because sorghum is a cross-pollinated crop with a high natural cross-pollination rate, existing breeding techniques cannot effectively solve the problem of biological contamination, making it difficult to stably promote and apply superior varieties.

[0004] Therefore, there is an urgent need for a method for breeding sorghum varieties for liquor that integrates multidisciplinary technologies, adapts to the process requirements of Maotai-flavor liquor, and takes into account both variety selection and breeding of superior varieties. This method aims to cultivate high-quality, high-yield, and disease-resistant new varieties of sorghum specifically for liquor production, formulate scientific breeding procedures, ensure stable yield and quality of sorghum for liquor production, meet the raw material needs of brewing enterprises, and promote the coordinated development of agriculture and the liquor industry. Summary of the Invention

[0005] This invention provides a method for breeding sorghum varieties for brewing, applicable to the raw material supply links of agricultural production and the liquor industry. It effectively solves the problems of aggravated pests and diseases, mixed varieties and degeneration, decreased yield and quality deterioration caused by the long-term monoculture of mainstream sorghum varieties and lack of systematic purification and rejuvenation in the current production of liquor sorghum, thus promoting increased agricultural production and efficiency and high-quality development of the sauce-flavored liquor industry.

[0006] This invention provides a method for breeding sorghum varieties for brewing, comprising: S1. Based on the requirements of the brewing process of Maotai-flavor liquor and the problem of variety degradation in production, determine the targets for quality, yield, resistance and adaptability. Then, according to the targets, collect and innovate sorghum germplasm through traditional and modern breeding techniques to produce new line materials that are suitable for the targets. S2. The agronomic traits and basic physicochemical indicators of the new strains are evaluated. After screening out the qualified strains, metabolomics analysis and differential metabolite screening are performed by UPLC-MS / MS to obtain candidate strains with both basic trait advantages and metabolic advantages. S3. The selected candidate strains are tested in multiple ecological zones to verify their high yield, stable yield and stress resistance. Then, the ecologically adapted strains are subjected to precise quality re-inspection and small-scale brewing of Maotai-flavor liquor to obtain high-quality special liquor sorghum strains that meet the process requirements and are suitable for actual production.

[0007] Furthermore, S1 specifically includes: Based on the technological requirements of nine distillations, eight fermentations, and seven extractions for Maotai-flavor liquor, and considering the issue of Hongyingzi variety degradation during production, S101 sets targets for quality, yield, resistance, and adaptability, specifically including: Quality: Glutinous sorghum with a starch content of ≥65% and amylopectin accounting for ≥88% of the total starch, tannin content of 1.5%-2.0%, thousand-grain weight of 16-22g, and protein and fat content that meet brewing standards; Yield: ≥5% increase compared to control varieties; single ear grain weight and thousand-grain weight are greater than conventional varieties; Resistance: Enhances disease resistance and stress tolerance; Adaptability: It can be planted in multiple ecological zones; S102. Collect local varieties, conventional varieties and hybrid varieties to establish a germplasm resource bank. Based on the germplasm resource bank, use a combination of traditional breeding and modern biotechnology to carry out germplasm innovation for the target traits determined in step S201 and adapt new line materials to the target.

[0008] Further, S102 specifically includes: Local varieties, conventional varieties, and hybrid varieties were collected, and a germplasm resource bank was constructed according to germplasm type, core trait parameters, and native habitat information. The core trait parameters include yield-related, quality-related, resistance-related, and adaptability-related parameters. Based on the breeding objectives of brewing sorghum, screening thresholds were set, specifically: yield advantage, quality suitability, resistance foundation, and plant type advantage. Germplasm files were retrieved from the germplasm resource bank to initially screen out germplasm that met at least three screening thresholds. Field planting verification was then conducted on the initially screened germplasm to finally determine the core parents, including high-yield parents, process-suitable parents, and disease-resistant parents. Germplasm that did not meet the requirements for brewing in terms of key indicators was eliminated. Based on the screened core parents, hybrid combinations with complementary traits were designed. Under isolation conditions, artificial emasculation and pollination were carried out during the peak flowering period of sorghum. The plants were then bagged and isolated until the grains matured. F1 generation hybrid seeds were harvested and planted. F2 generation was obtained through self-pollination. Individual plants meeting the target traits were selected using the single-plant selection method. Using the off-site, off-season generation technology, self-pollination and purification were accelerated to F4-F7 generations to obtain intermediate lines with stable traits. These intermediate lines must meet the following requirements for two consecutive generations: a coefficient of variation of single ear grain weight ≤5% and a coefficient of variation of tannin content ≤8%. From the core parents selected, germplasm with a single indicator to be optimized was selected, and its gene mutation was induced by physical mutagenesis, chemical mutagenesis or air breeding. The seeds after mutagenesis treatment were planted and self-pollinated in the M1 generation to obtain the M2 generation. Through field phenotypic observation and laboratory testing, the variant lines with optimized indicators were screened and added to the innovative variant library of the germplasm resource bank. To target key traits of brewing sorghum, we identified and validated associated molecular markers. For the obtained stable intermediate lines and variant lines, we extracted genomic DNA from the leaves and amplified the target marker fragments using PCR technology. The amplification products were detected by agarose gel electrophoresis or capillary electrophoresis. We screened for lines that simultaneously carried the dwarf marker S1, the anthracnose resistance marker R1, and the high amylopectin marker A1. We removed lines that did not carry or carried only one target marker to obtain candidate lines with precise targeting. Candidate strains were artificially inoculated to identify disease resistance. Standardized planting conditions were provided for the candidate strains. During the grain maturity period, the starch content, amylopectin ratio, tannin content, protein content, and fat content of the candidate strains were measured. Strains that met all the physicochemical requirements for brewing were screened. Based on the results of disease resistance identification and physicochemical test results, new strains with the target traits were determined.

[0009] Furthermore, S2 specifically includes: S201. Select 10 uniformly growing samples from the new strain materials, measure the agronomic traits of plant height, single ear grain weight, and thousand-grain weight, and at the same time test the basic physicochemical indicators of starch, amylose, protein, fat, and tannins, and screen out the strains that have agronomic traits exceeding the set values ​​and whose basic physicochemical indicators meet the requirements of S1. S202. Take the seeds of the pre-evaluated qualified lines, treat them with the extract, freeze grind them, and extract them with low-temperature ultrasonication. Then, use a UPLC-MS / MS system with dual chromatographic columns for separation and detection. Obtain metabolite data by matching the MJDB-PM database with Progenesis QI. Then, screen for differential metabolites by PCA and OPLS-DA analysis to obtain candidate lines with both basic trait advantages and metabolic advantages.

[0010] Furthermore, S3 specifically includes: S301. Selected candidate strains will be tested in regional and production trials in multiple typical ecological zones. Each test site will be replicated three times, with the local main cultivated variety as a control. The results will be observed for 2-3 consecutive growth cycles, and data on yield, yield stability, and stress resistance will be recorded. Ecologically suitable strains that show stable performance in each ecological zone will be selected. S302. After accurately testing the core quality indicators of starch, amylopectin, and tannins of the ecologically adapted strains again and confirming that they meet the process requirements of S1, conduct small-scale brewing according to the standard process of sauce-flavored liquor, determine the alcohol yield and analyze the quality of the liquor, and screen out high-quality special liquor sorghum strains.

[0011] Furthermore, after S3, it also includes: S4. Perform DUS testing on the high-quality special-purpose sorghum strain for liquor production, and record the specificity, consistency, and stability data. At the same time, use PCR technology to perform non-GMO detection, and record the target sorghum strain that passes both tests.

[0012] Furthermore, after S4, it also includes: S5. Using the target sorghum line obtained in S4 as the parent, establish a superior seed breeding base in the isolation area to study the seed development mechanism, nutrient accumulation law, the impact of harvest period on vigor, and data on changes in storage vigor. Formulate operating procedures for the breeding of sorghum varieties for liquor production, so as to clarify the standards for sowing, water and fertilizer, impurity removal, harvesting, and storage, and breed qualified seeds with a purity of ≥98% and a germination rate of ≥85%.

[0013] Furthermore, after S5, it also includes: S6. Establish seed breeding bases in major liquor-producing areas according to the operating procedures for breeding liquor sorghum varieties, transfer planting techniques through farmer training, monitor farmer income and raw material supply data of liquor enterprises, evaluate the actual effects of new varieties on agricultural production and raw material security of the liquor industry, and feed the evaluation data back to S1 to verify the achievement of the above objectives.

[0014] The beneficial effects of this invention are as follows: This invention integrates traditional breeding with modern biotechnology and combines multidisciplinary research efforts to cultivate new sorghum varieties for liquor production that possess high quality, high yield, and disease resistance. Combined with regional and production trials under different ecological conditions, it can screen suitable varieties for planting in regions with varying ecological types, expanding the planting area of ​​sorghum for liquor production. Simultaneously, through targeted quality testing and compatibility tests with the Maotai-flavor liquor brewing process, it ensures that the new varieties meet the specific requirements of the nine distillations, eight fermentations, and seven extraction processes for Maotai-flavor liquor, helping to maintain and enhance the brand competitiveness of Maotai-flavor liquor. Furthermore, by conducting DUS testing and non-GMO testing, the compliance of the new varieties is guaranteed, effectively solving the biological contamination problem caused by cross-pollination of sorghum, achieving increased yield, income, and efficiency of sorghum production, and providing a stable and high-quality raw material supply for brewing enterprises to meet the development needs of the liquor industry. Detailed Implementation

[0015] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0016] This invention provides a method for breeding sorghum varieties for brewing, comprising: S1. Based on the requirements of the Maotai-flavor liquor brewing process and the problem of variety degradation in production, determine the targets for quality, yield, resistance, and adaptability. Then, according to the targets, collect and innovate sorghum germplasm through traditional and modern breeding techniques to produce new line materials that are suitable for the targets.

[0017] Based on the technological requirements of nine distillations, eight fermentations, and seven extractions for Maotai-flavor liquor (such as ≥65% glutinous sorghum starch, ≥88% amylopectin, 1.5%-2.0% tannins, and 16-22g per thousand grains), and addressing the issue of varietal degeneration in the Hongyingzi variety (increased pests and diseases, and low alcohol yield), S101 sets targets for quality, yield, resistance, and adaptability, specifically including: a. Quality: Meets the requirements of the "nine distillations, eight fermentations, and seven extractions" process for Maotai-flavor liquor. Specifically, it uses glutinous sorghum (starch content ≥65%, amylopectin accounts for ≥88% of total starch), tannin content 1.5%-2.0%, thousand-grain weight 16-22g, and protein (7%-9%) and fat (moderate, avoiding excessive acid production) content that meet brewing standards.

[0018] b. Yield: ≥5% increase compared to control varieties (such as Hongyingzi), with significantly better single-ear grain weight and thousand-grain weight than conventional varieties (refer to the advantages of HS08 with single-ear grain weight of 87.73g and thousand-grain weight of 21.13g).

[0019] c. Resistance: Enhance disease resistance (such as resistance to anthracnose) and stress resistance, reduce pesticide use, and lower production costs.

[0020] d. Adaptability: It has wide adaptability and can be stably planted in multiple ecological zones (such as major liquor-producing areas such as southern Sichuan and Guizhou).

[0021] S102. Collect local varieties, conventional varieties, and hybrid varieties to establish a germplasm resource bank. Based on the germplasm resource bank, use a combination of traditional breeding (hybridization, mutation, and air breeding) and modern biotechnology (molecular marker-assisted selection, off-site and off-season generation) to innovate germplasm for the target traits (such as dwarf stalks, large ears, early maturity, and disease resistance) determined in step S201, and develop new lines adapted to the targets. Specifically, (1) Collect local varieties (such as HS08, Niuweizi, Yanggaoliang, Qipiye, and Maweigaoliang), conventional varieties (such as Guojiaohong No. 1, Hongyingzi, Langnuohong No. 19, Luzhouhong No. 1, and Yinuohong No. 4) and hybrid varieties (such as Qiza 104, Jinza 22, Jiza 210, and Fengza 4), and construct a germplasm resource bank according to germplasm type (local variety / conventional variety / hybrid variety) - core trait parameters - native habitat information. Among them, the core trait parameters include yield-related (historical single ear grain weight, thousand-grain weight), quality-related (historical starch content, amylopectin ratio, tannin content, protein content, and fat content), resistance-related (historical disease and pest resistance records, stress resistance evaluation), and adaptability-related (native planting area, ecological suitability).

[0022] (2) Based on the breeding objectives of sorghum for brewing (short stalk, large ears, early maturity, disease resistance, and suitability for sauce-flavored liquor production processes), screening thresholds were set, specifically: yield advantage (single ear grain weight ≥75g, thousand-grain weight 18-22g), quality suitability (starch content ≥65%, amylopectin percentage ≥88%, tannins 1.5%-2.0%, protein 7%-9%, fat 3.5%-4.5%), resistance foundation (historical disease and pest resistance level ≥medium resistance), and plant type advantage (plant type...). (Height ≤ 250cm), retrieve germplasm files through the germplasm resource bank described in (1), initially screen out germplasm that meets at least 3 screening thresholds, and verify the initial screening germplasm by field planting, and finally determine the core parents, including high-yield parents, process-adapted parents, and disease-resistant parents, and remove germplasm that does not meet the requirements of brewing in terms of key indicators, specifically tannin > 3% or < 1.2%, starch < 62%, thousand-grain weight < 16g, and germplasm with a historical disease resistance level of susceptible or below.

[0023] (3) Based on the core parents screened in (2), design hybrid combinations with complementary traits, such as: high-yielding parent (HS08, single ear grain weight 87.73g) × short-stalked disease-resistant parent (such as local short-stalked varieties, plant height ≤220cm, resistant to anthracnose), process-adaptable parent (Hongyingzi, tannin 1.8%) × large ear parent (such as Luzhou Red No. 1, single ear grain weight 78g).

[0024] Under isolation conditions (spatial isolation distance ≥300m to avoid cross-pollination), artificial emasculation pollination was carried out during the peak flowering period of sorghum. Specifically, the female plant's open flowers on the same day were selected, the stamens were removed, fresh pollen from the male plant was collected and applied to the stigma of the female plant, and the plant was bagged and isolated until the grains matured. F1 generation hybrid seeds were then harvested.

[0025] F1 generation seeds were planted and self-pollinated to obtain F2 generation. Single plants meeting the target traits (plant height 220-240cm, single ear grain weight ≥80g, tannin 1.5%-2.0%) were selected using the single plant selection method. Using the off-site off-season generation technology, self-pollination and purification were accelerated to F4-F7 generation to obtain intermediate lines with stable traits. The intermediate lines must meet the following requirements for two consecutive generations: single ear grain weight variation coefficient ≤5% and tannin content variation coefficient ≤8%.

[0026] (4) From the core parents screened in (2), select germplasm with excellent basic traits but single indicators that need to be optimized, such as Hongyingzi (optimization direction: increase yield and enhance lodging resistance) and Langnuohong 19 (optimization direction: shorten growth period and increase flavonoid content).

[0027] Mutations were induced in the genes by physical mutagenesis, chemical mutagenesis, or air breeding. The mutagenized seeds were planted and self-crossed to obtain the M2 generation. Through field phenotypic observation (growth period, plant height, disease resistance) and laboratory testing (yield, physicochemical indicators), mutant lines with optimized indicators (such as a 5-7 day shortened growth period, a 1-level improvement in lodging resistance, and a more than 10% increase in single ear grain weight) were screened and added to the innovative variant library of the germplasm resource bank.

[0028] a. Physical mutagenesis: Seeds were irradiated with 60Co-γ rays at a dose of 150-200 Gy and a dose rate of 1 Gy / min. After irradiation, the seeds were placed in a constant temperature environment of 25℃ for 24 hours to recover.

[0029] b. Chemical mutagenesis: Soak seeds in 0.1%-0.2% ethyl methanesulfonate (EMS) solution at 25℃ for 8-12 hours. After soaking, rinse with sterile water 3-5 times and air dry for later use.

[0030] c. Aerospace breeding: Seeds are carried into low Earth orbit on spacecraft, with an on-orbit flight time of ≥72 hours. Gene mutations are induced using cosmic rays and microgravity environment. After returning to Earth, the seeds are planted on the ground.

[0031] (5) For key target traits of brewing sorghum, we explored and verified the associated molecular markers, specifically: dwarf stalk trait (associated with SNP marker S1, located on chromosome 3), anthrax resistance trait (associated with SSR marker R1, located on chromosome 7), and high amylopectin trait (associated with InDel marker A1, located on chromosome 5).

[0032] Genomic DNA was extracted from the leaves of the stable intermediate line obtained in (3) and the mutant line obtained in (4), and the target marker fragment was amplified by PCR. The PCR reaction system consisted of 10 μL of 2×Taq Master Mix, 0.5 μL each of upstream and downstream primers (10 μmol / L), 2 μL of DNA template, and 7 μL of ddH2O. The reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55-60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and 72℃ final extension for 10 min.

[0033] The amplification products were detected by agarose gel electrophoresis or capillary electrophoresis. Lines carrying the dwarf marker S1, the anthracnose resistance marker R1, and the high branched-chain starch marker A1 were screened. Lines that did not carry or carried only one target marker were removed to obtain candidate lines for precise targeting.

[0034] (6) Artificial inoculation was performed to identify the disease resistance of candidate lines. Specifically, anthracnose fungus (concentration 1×106 cells / mL) was inoculated during the seedling stage. After 21 days, the disease level was investigated and lines with a disease level ≤2 (0: no lesions; 1: lesions occupy ≤5% of the leaf area; 2: lesions occupy 6%-15% of the leaf area) were selected.

[0035] Standardized planting conditions were provided for candidate lines, specifically: the amount of basal fertilizer applied (300 kg / hm² of N-P2O5-K2O=15-15-15 compound fertilizer) and the timing of topdressing (150 kg / hm² of urea applied at the jointing stage) to ensure normal expression of line traits.

[0036] During the grain maturity period, the starch content, amylopectin ratio, tannin content, protein content, and fat content of candidate strains are measured. Strains that meet all the physicochemical requirements for brewing are screened. Based on the results of disease resistance identification and physicochemical test results, new strains with the target traits (short stalk, large ears, high yield, disease resistance, and process suitability) are determined.

[0037] S2. The agronomic traits and basic physicochemical indicators of the new strains are evaluated. After screening out the qualified strains, metabolomics analysis and differential metabolite screening are performed by UPLC-MS / MS to obtain candidate strains that have both basic trait advantages and metabolic advantages.

[0038] S201. Select 10 uniformly growing samples from the new line materials described in S102(6), and measure the agronomic traits of plant height, single ear grain weight, and thousand-grain weight. At the same time, test the basic physicochemical indicators of starch, amylose, protein, fat, and tannins. Screen out the preliminary qualified lines whose agronomic traits exceed the set values ​​(such as single ear grain weight ≥80g, thousand-grain weight ≥20g) and whose basic physicochemical indicators meet the requirements of S1 (starch ≥65%, tannin 1.5%-2.0%). S202. Seeds of the pre-evaluated qualified lines were treated with extract, freeze-ground, and extracted using low-temperature ultrasonication. The extracted metabolites were then separated and detected using a UPLC-MS / MS system with dual chromatographic columns. Metabolite data were obtained by matching the MJDB-PM database with the Progenesis QI. Differential metabolites were then screened by PCA and OPLS-DA analysis (threshold VIP > 2, P < 0.05, Fold change = 1). Flavonoids (quercetin, kaempferol), terpenes, and other substances related to resistance and flavor were given special attention. Upregulated pathways such as flavonoid biosynthesis were confirmed by KEGG pathway enrichment to obtain candidate lines with both basic trait advantages and metabolic advantages.

[0039] S3. The selected candidate strains are tested in multiple ecological zones to verify their high yield, stable yield and stress resistance. Then, the ecologically adapted strains are subjected to precise quality re-inspection and small-scale brewing of Maotai-flavor liquor to obtain high-quality special liquor sorghum strains that meet the process requirements and are suitable for actual production.

[0040] S301. Selected candidate lines will be used for regional and production trials in multiple typical ecological zones. Each trial site will have three replicates, with the local main cultivated variety (such as Hongyingzi) as a control. The trials will be observed for 2-3 consecutive growth cycles, and data on yield (yield per mu, number of grains per ear), yield stability (annual yield fluctuation), and stress resistance (resistance to diseases and pests, resistance to lodging) will be recorded. Ecologically suitable lines that show stable performance in each ecological zone (yield fluctuation ≤10%, disease resistance rate ≥80%) will be selected. S302. The ecologically adapted strains are tested again for core quality indicators such as starch (≥65%), amylopectin (≥88%), and tannins (1.5%-2.0%). After confirming that they meet the process requirements of S1, small-scale brewing is carried out according to the standard process of Maotai-flavor liquor. The alcohol yield is measured (target ≥45%), and the quality of the liquor is analyzed (flavor substances such as furan, pyrazine content, and fusel oil content). High-quality special-purpose sorghum strains for liquor are selected.

[0041] S4. Perform DUS testing on the high-quality special-purpose sorghum strains for liquor production, and record the data on specificity (distinguishing traits from existing varieties), uniformity (individual differences within the population ≤5%), and stability (overlap of traits between different generations ≥95%). At the same time, use PCR technology to perform non-GMO testing, and record the target sorghum strains that pass both tests (DUS compliance and no GMO components).

[0042] S5. Using the target sorghum line obtained in S4 as the parent, establish a superior seed breeding base in the isolation area to study the seed development mechanism, nutrient accumulation pattern, the impact of harvest period on vigor (e.g., the optimal harvest period is when the grain moisture content is 12%-14%), and storage vigor change data. Formulate operating procedures for the breeding of sorghum varieties for liquor production, so as to clarify the standards for sowing, water and fertilizer, impurity removal, harvesting, and storage, and breed qualified seeds with a purity of ≥98% and a germination rate of ≥85%.

[0043] S6. Establish seed breeding bases in major liquor-producing areas according to the operating procedures for breeding liquor sorghum varieties. Transmit planting techniques through farmer training, monitor farmer income (yield increase, pesticide cost reduction rate), and raw material supply data for liquor enterprises (quality stability, supply volume). Evaluate the actual effects of new varieties on agricultural production increase (target yield increase ≥5%) and raw material security for the liquor industry, and feed the evaluation data back to S1 to verify the achievement of the stated objectives, forming a closed loop of selection-breeding-promotion-feedback.

[0044] This invention integrates traditional breeding with modern biotechnology and combines multidisciplinary research efforts to cultivate new sorghum varieties for liquor production that possess high quality, high yield, and disease resistance. Combined with regional and production trials under different ecological conditions, it can screen suitable varieties for planting in regions with varying ecological types, expanding the planting area of ​​sorghum for liquor production. Simultaneously, through targeted quality testing and compatibility tests with the Maotai-flavor liquor brewing process, it ensures that the new varieties meet the specific requirements of the nine distillations, eight fermentations, and seven extraction processes for Maotai-flavor liquor, helping to maintain and enhance the brand competitiveness of Maotai-flavor liquor. Furthermore, by conducting DUS testing and non-GMO testing, the compliance of the new varieties is guaranteed, effectively solving the biological contamination problem caused by cross-pollination of sorghum, achieving increased yield, income, and efficiency of sorghum production, and providing a stable and high-quality raw material supply for brewing enterprises to meet the development needs of the liquor industry.

[0045] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, apparatus, article, or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, apparatus, article, or method. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, apparatus, article, or method that includes that element.

[0046] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for breeding sorghum varieties for brewing, characterized in that, include: S1. Based on the requirements of the brewing process of Maotai-flavor liquor and the problem of variety degradation in production, determine the targets for quality, yield, resistance and adaptability. Then, according to the targets, collect and innovate sorghum germplasm through traditional and modern breeding techniques to produce new line materials that are suitable for the targets. S2. The agronomic traits and basic physicochemical indicators of the new strains are evaluated. After screening out the qualified strains, metabolomics analysis and differential metabolite screening are performed by UPLC-MS / MS to obtain candidate strains with both basic trait advantages and metabolic advantages. S3. The selected candidate strains are tested in multiple ecological zones to verify their high yield, stable yield and stress resistance. Then, the ecologically adapted strains are subjected to precise quality re-inspection and small-scale brewing of Maotai-flavor liquor to obtain high-quality special liquor sorghum strains that meet the process requirements and are suitable for actual production.

2. The method for breeding sorghum varieties for brewing according to claim 1, characterized in that, S1 specifically includes: Based on the technological requirements of nine distillations, eight fermentations, and seven extractions for Maotai-flavor liquor, and considering the issue of Hongyingzi variety degradation during production, S101 sets targets for quality, yield, resistance, and adaptability, specifically including: Quality: Glutinous sorghum with a starch content of ≥65% and amylopectin accounting for ≥88% of the total starch, tannin content of 1.5%-2.0%, thousand-grain weight of 16-22g, and protein and fat content that meet brewing standards; Yield: ≥5% increase compared to control varieties; single ear grain weight and thousand-grain weight are greater than conventional varieties; Resistance: Enhances disease resistance and stress tolerance; Adaptability: It can be planted in multiple ecological zones; S102. Collect local varieties, conventional varieties and hybrid varieties to establish a germplasm resource bank. Based on the germplasm resource bank, use a combination of traditional breeding and modern biotechnology to carry out germplasm innovation for the target traits determined in step S201 and adapt new line materials to the target.

3. The method for breeding sorghum varieties for brewing according to claim 2, characterized in that, S102 specifically includes: Local varieties, conventional varieties, and hybrid varieties were collected, and a germplasm resource bank was constructed according to germplasm type, core trait parameters, and native habitat information. The core trait parameters include yield-related, quality-related, resistance-related, and adaptability-related parameters. Based on the breeding objectives of brewing sorghum, screening thresholds were set, specifically: yield advantage, quality suitability, resistance foundation, and plant type advantage. Germplasm files were retrieved from the germplasm resource bank to initially screen out germplasm that met at least three screening thresholds. Field planting verification was then conducted on the initially screened germplasm to finally determine the core parents, including high-yield parents, process-suitable parents, and disease-resistant parents. Germplasm that did not meet the requirements for brewing in terms of key indicators was eliminated. Based on the screened core parents, hybrid combinations with complementary traits were designed. Under isolation conditions, artificial emasculation and pollination were carried out during the peak flowering period of sorghum. The plants were then bagged and isolated until the grains matured. F1 generation hybrid seeds were harvested and planted. F2 generation was obtained through self-pollination. Individual plants meeting the target traits were selected using the single-plant selection method. Using the off-site, off-season generation technology, self-pollination and purification were accelerated to F4-F7 generations to obtain intermediate lines with stable traits. These intermediate lines must meet the following requirements for two consecutive generations: a coefficient of variation of single ear grain weight ≤5% and a coefficient of variation of tannin content ≤8%. From the core parents selected, germplasm with a single indicator to be optimized was selected, and its gene mutation was induced by physical mutagenesis, chemical mutagenesis or air breeding. The seeds after mutagenesis treatment were planted and self-pollinated in the M1 generation to obtain the M2 generation. Through field phenotypic observation and laboratory testing, the variant lines with optimized indicators were screened and added to the innovative variant library of the germplasm resource bank. To target key traits of brewing sorghum, we identified and validated associated molecular markers. For the obtained stable intermediate lines and variant lines, we extracted genomic DNA from the leaves and amplified the target marker fragments using PCR technology. The amplification products were detected by agarose gel electrophoresis or capillary electrophoresis. We screened for lines that simultaneously carried the dwarf marker S1, the anthracnose resistance marker R1, and the high amylopectin marker A1. We removed lines that did not carry or carried only one target marker to obtain candidate lines with precise targeting. Candidate strains were artificially inoculated to identify disease resistance. Standardized planting conditions were provided for the candidate strains. During the grain maturity period, the starch content, amylopectin ratio, tannin content, protein content, and fat content of the candidate strains were measured. Strains that met all the physicochemical indicators for brewing were screened. Based on the results of disease resistance identification and physicochemical indicator testing, new strains with the target traits were determined.

4. The method for breeding sorghum varieties for brewing according to claim 1, characterized in that, S2 specifically includes: S201. Select 10 uniformly growing samples from the new strain materials, measure the agronomic traits of plant height, single ear grain weight, and thousand-grain weight, and at the same time test the basic physicochemical indicators of starch, amylose, protein, fat, and tannins, and screen out the strains that have agronomic traits exceeding the set values ​​and whose basic physicochemical indicators meet the requirements of S1. S202. Take the seeds of the pre-evaluated qualified lines, treat them with the extract, freeze grind them, and extract them with low-temperature ultrasonication. Then, use a UPLC-MS / MS system with dual chromatographic columns for separation and detection. Obtain metabolite data by matching the MJDB-PM database with Progenesis QI. Then, screen for differential metabolites by PCA and OPLS-DA analysis to obtain candidate lines with both basic trait advantages and metabolic advantages.

5. The method for breeding sorghum varieties for brewing according to claim 1, characterized in that, S3 specifically includes: S301. Selected candidate strains will be tested in regional and production trials in multiple typical ecological zones. Each test site will be replicated three times, with the local main cultivated variety as a control. The results will be observed for 2-3 consecutive growth cycles, and data on yield, yield stability, and stress resistance will be recorded. Ecologically suitable strains that show stable performance in each ecological zone will be selected. S302. After accurately testing the core quality indicators of starch, amylopectin, and tannins of the ecologically adapted strains again and confirming that they meet the process requirements of S1, conduct small-scale brewing according to the standard process of sauce-flavored liquor, determine the alcohol yield and analyze the quality of the liquor, and screen out high-quality special liquor sorghum strains.

6. The method for breeding sorghum varieties for brewing according to claim 1, characterized in that, Following S3, the following also includes: S4. Perform DUS testing on the high-quality special-purpose sorghum strain for liquor production, and record the specificity, consistency, and stability data. At the same time, use PCR technology to perform non-GMO detection, and record the target sorghum strain that passes both tests.

7. The method for breeding sorghum varieties for brewing according to claim 6, characterized in that, Following S4, the following is also included: S5. Using the target sorghum line obtained in S4 as the parent, establish a superior seed breeding base in the isolation area to study the seed development mechanism, nutrient accumulation law, the impact of harvest period on vigor, and data on changes in storage vigor. Formulate operating procedures for the breeding of sorghum varieties for liquor production, so as to clarify the standards for sowing, water and fertilizer, impurity removal, harvesting, and storage, and breed qualified seeds with a purity of ≥98% and a germination rate of ≥85%.

8. The method for breeding sorghum varieties for brewing according to claim 7, characterized in that, Following S5, the following is also included: S6. Establish seed breeding bases in major liquor-producing areas according to the operating procedures for breeding liquor sorghum varieties, transfer planting techniques through farmer training, monitor farmer income and raw material supply data of liquor enterprises, evaluate the actual effects of new varieties on agricultural production and raw material security of the liquor industry, and feed the evaluation data back to S1 to verify the achievement of the above objectives.