A high-polymer selenium soybean germplasm breeding method

Abstract

This invention provides a method for breeding high-selenium soybean germplasm, comprising: continuously planting wild and cultivated soybean germplasm in selenium-rich areas with soil selenium content of 0.4-0.5 mg / kg for 2-3 years; screening for high-selenium donors with grain selenium content greater than 0.35 mg / kg and superior agronomical recipient parents with yield greater than or equal to 150 kg / mu and protein content greater than or equal to 40%; obtaining first-generation populations through parental hybridization and constructing second-generation segregating populations through self-pollination; measuring root activity at flowering stage and the ratio of stem to grain selenium content at grain filling stage to establish quantitative screening thresholds; further, performing a three-step progressive and precise screening of root activity, antioxidant enzyme activity, and grain selenium content on the second-generation and subsequent self-pollinating generations; purifying through multiple generations from three to five, and identifying in different ecological regions, ultimately selecting soybean germplasm with a stable grain selenium content greater than 0.35 mg / kg under various environments. This method achieves efficient aggregation of high-selenium trait and superior agronomic traits, with stable and reliable breeding results, wide adaptability, and value for large-scale promotion.

Description

Technical Field

[0001] This invention relates to the field of soybean variety breeding technology, and more specifically, to a method for breeding high-selenium soybean germplasm. Background Technology

[0002] Selenium is an essential trace element for the human body, participating in various physiological processes such as immune regulation and antioxidation, and playing an irreplaceable role in maintaining human health. As a major global food crop and source of plant protein, the selenium content of soybean seeds directly relates to dietary selenium intake. Developing high-selenium soybean germplasm is of significant practical importance for improving residents' selenium nutrition status and promoting the development of selenium-enriched agricultural products.

[0003] Currently, the breeding technology for high-selenium soybeans is still imperfect, and there are many limitations in practical applications. The screening results for high-selenium soybean materials are unstable, with low consistency in trait expression under different environments, making it difficult to obtain stable and high-quality germplasm. The bred soybean materials generally suffer from the problem of not being able to simultaneously achieve high selenium content with agronomical traits such as high yield and quality, resulting in poor overall production performance. Some bred materials exhibit poor environmental adaptability and insufficient trait stability during promotion and planting, making it difficult to meet the requirements of large-scale production applications. Overall, the current level of high-selenium soybean breeding is limited, and stable, high-quality, and widely adaptable high-selenium soybean germplasm resources are relatively scarce, making it difficult to support the high-quality development of the industry.

[0004] Therefore, there is an urgent need for a breeding method for high-selenium soybean germplasm to solve the problems in the existing technology. Summary of the Invention

[0005] The main objective of this invention is to provide a method for breeding high-selenium soybean germplasm, so as to at least solve the problems of poor breeding stability, unstable trait inheritance, difficulty in achieving both high-selenium traits and excellent agronomic traits, and insufficient environmental adaptability in the prior art.

[0006] To achieve the above objectives, the present invention provides a method for breeding high-selenium soybean germplasm, such as... Figure 1 As shown, it includes:

[0007] Step 1: Plant soybean germplasm resources in selenium-rich areas with soil selenium content of 0.4-0.5 mg / kg for 2-3 years. The sources of soybean germplasm resources include wild soybean germplasm and cultivated soybean germplasm. Screen high selenium donor materials with grain selenium content greater than 0.35 mg / kg and agronomically excellent recipient materials with a yield greater than or equal to 150 kg / mu and a protein content greater than or equal to 40%.

[0008] Step 2: Using the high-selenium donor material as the male parent and the agronomically superior recipient material as the female parent, hybridize to obtain the first generation, and self-pollinate to obtain the second generation genetic segregating population. Measure the ratio of root activity at flowering time to stem selenium content and grain selenium content at the grain-filling stage in the second generation genetic segregating population. Based on the ratio of root activity at flowering time to stem selenium content and grain selenium content at the grain-filling stage corresponding to individual plants with grain selenium content greater than 0.35 mg / kg, determine the threshold for root activity at flowering time and the threshold for the ratio of stem selenium content and grain selenium content at the grain-filling stage.

[0009] Step 3: Perform a three-step screening on the second-generation genetic segregation population: First, pre-screen individual plants based on the root vigor threshold at flowering time and the stem-to-seed selenium content ratio threshold at the grain-filling stage; Second, measure the antioxidant enzyme activity of the pre-screened individual plants at the seedling stage, and retain individual plants with antioxidant enzyme activity not lower than the activity threshold; Third, from the individual plants retained in the second step, screen for individual plants with a mature grain selenium content greater than 0.35 mg / kg to obtain superior hybrid plants;

[0010] Step 4: Self-pollinate the hybrid superior plants to obtain three generations, and perform the three-step screening in Step 3 on the three generations; self-pollinate the single plants obtained from the three generations to obtain four generations, and perform the three-step screening in Step 3 on the four generations; then self-pollinate the single plants obtained from the four generations to obtain five generations, and perform the three-step screening in Step 3 on the five generations; conduct field identification of the lines obtained from the five generations in different ecological regions, and select soybean germplasm with a grain selenium content greater than 0.35 mg / kg when planted in different ecological regions.

[0011] Optionally, the drought resistance damage rate and cold resistance damage rate of the wild soybean germplasm described in step 1 are ≤30%.

[0012] Optionally, the root activity during the flowering period in step 2 is determined by the TTC method, and the selenium content in the stems and grains is determined by atomic fluorescence spectrometry.

[0013] Optionally, the antioxidant enzyme activity in step 3 during the seedling stage is superoxide dismutase activity.

[0014] Optionally, the antioxidant enzyme activity described in step 3 is determined using the nitroblue tetrazolium method.

[0015] Optionally, during the determination using the nitroblue tetrazolium method, the reaction temperature is controlled at 20-30℃ and the reaction time is controlled at 10-20 min.

[0016] Optionally, step 3, which involves pre-screening individual plants based on the root vigor threshold during flowering and the ratio threshold of stem selenium content to grain selenium content during grain filling, specifically includes:

[0017] A dual-weighted scoring method was used to evaluate root activity during flowering and the ratio of stem to grain selenium content during grain filling. The weighting coefficient for the ratio of stem to grain selenium content during grain filling was 0.7, and the weighting coefficient for root activity during flowering was 0.3. Individual plants with root activity during flowering not lower than the threshold value and a ratio of stem to grain selenium content during grain filling lower than the threshold value, and whose overall score reached the preset threshold value, were retained.

[0018] Optionally, in step 4, the field identification adopts simultaneous planting in multiple selenium-rich gradient areas. The different ecological areas include at least a high selenium area with soil selenium content of 0.4-0.5 mg / kg and a medium-high selenium area with soil selenium content of 0.2-0.4 mg / kg, and the planting density in each ecological area is 10,000-15,000 plants / mu.

[0019] Optionally, during the screening process in steps 3 and 4, individual plants with a selenium content greater than 0.35 mg / kg but a protein content less than 40% are excluded.

[0020] Optionally, the selenium content of the grains is detected when the grains are fully mature and naturally air-dried to a moisture content of 12-14%.

[0021] This invention discloses a method for breeding high-selenium soybean germplasm, comprising: Step 1, planting soybean germplasm resources for 2-3 years in a selenium-rich area with soil selenium content of 0.4-0.5 mg / kg, wherein the soybean germplasm resources include wild soybean germplasm and cultivated soybean germplasm, screening for high-selenium donor materials with a seed selenium content greater than 0.35 mg / kg, and agronomically superior recipient materials with a yield greater than or equal to 150 kg / mu and a protein content greater than or equal to 40%; Step 2, hybridizing the high-selenium donor material as the male parent and the agronomically superior recipient material as the female parent to obtain the first generation, self-pollinating to obtain the second generation genetic segregating population, and measuring the ratio of root activity at flowering time to stem selenium content and seed selenium content at the pod-filling stage of the second generation genetic segregating population; determining the threshold of root activity at flowering time and the ratio of stem selenium content and seed selenium content at the pod-filling stage corresponding to individual plants with a seed selenium content greater than 0.35 mg / kg. Content ratio threshold; Step 3: Perform three-step screening on the second-generation genetic segregation population: Step 1: Pre-screen individual plants according to the root vitality threshold at flowering time and the stem-to-seed selenium content ratio threshold at the grain filling stage; Step 2: Measure the antioxidant enzyme activity of the pre-screened individual plants at the seedling stage, and retain individual plants with antioxidant enzyme activity not lower than the activity threshold; Step 3: From the individual plants retained in Step 2, screen individual plants with a selenium content greater than 0.35 mg / kg at maturity to obtain hybrid superior plants; Step 4: Self-pollinate the hybrid superior plants to obtain the third generation, and perform the three-step screening in Step 3 on the third generation; Self-pollinate the individual plants obtained from the third generation to obtain the fourth generation, and perform the three-step screening in Step 3 on the fourth generation; Self-pollinate the individual plants obtained from the fourth generation to obtain the fifth generation, and perform the three-step screening in Step 3 on the fifth generation; Field identification of the lines obtained from the fifth generation screening in different ecological regions, and select soybean germplasm with a selenium content greater than 0.35 mg / kg when planted in different ecological regions. By screening parents in a stable selenium-rich environment over a long period, constructing hybrid segregating populations and determining key physiological thresholds, and combining multi-generational progressive quantitative screening with multi-ecological region identification, precise breeding and stable inheritance of high selenium-accumulating traits were achieved. This effectively improved the selenium accumulation capacity and trait stability of soybeans, while also taking into account excellent agronomic traits such as high yield and high protein. This resulted in high selenium-accumulating soybean germplasm that is widely adaptable and can be promoted on a large scale. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 This is a flowchart of a breeding method for high-selenium soybean germplasm, which is optional according to an embodiment of the present invention. Detailed Implementation

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0025] This application provides a method for breeding high-selenium soybean germplasm, characterized by comprising:

[0026] Step 1: Plant soybean germplasm resources in selenium-rich areas with soil selenium content of 0.4-0.5 mg / kg for 2-3 years, and screen high selenium donor materials with grain selenium content greater than 0.35 mg / kg, and agronomically excellent recipient materials with yield greater than or equal to 150 kg / mu and protein content greater than or equal to 40%.

[0027] Step 2: Using the high-selenium donor material as the male parent and the agronomically superior recipient material as the female parent, hybridize to obtain the first generation, and self-pollinate to obtain the second generation genetic segregating population. Measure the ratio of root activity at flowering time to stem selenium content and grain selenium content at the grain-filling stage in the second generation genetic segregating population. Based on the ratio of root activity at flowering time to stem selenium content and grain selenium content at the grain-filling stage corresponding to individual plants with grain selenium content greater than 0.35 mg / kg, determine the threshold for root activity at flowering time and the threshold for the ratio of stem selenium content and grain selenium content at the grain-filling stage.

[0028] Step 3: Perform a three-step screening on the second-generation genetic segregation population: First, pre-screen individual plants based on the root vigor threshold at flowering time and the stem-to-seed selenium content ratio threshold at the grain-filling stage; Second, measure the antioxidant enzyme activity of the pre-screened individual plants at the seedling stage, and retain individual plants with antioxidant enzyme activity not lower than the activity threshold; Third, from the individual plants retained in the second step, screen for individual plants with a mature grain selenium content greater than 0.35 mg / kg to obtain superior hybrid plants;

[0029] Step 4: Self-pollinate the hybrid superior plants to obtain three generations, and perform the three-step screening in Step 3 on the three generations; self-pollinate the single plants obtained from the three generations to obtain four generations, and perform the three-step screening in Step 3 on the four generations; then self-pollinate the single plants obtained from the four generations to obtain five generations, and perform the three-step screening in Step 3 on the five generations; conduct field identification of the lines obtained from the five generations in different ecological regions, and select soybean germplasm with a grain selenium content greater than 0.35 mg / kg when planted in different ecological regions.

[0030] Specifically, in step 1, a selenium-rich area with soil selenium content strictly controlled at 0.4-0.5 mg / kg is selected. This area provides a stable selenium supply for soybeans, creating a suitable environment for screening materials with high selenium accumulation capacity. In this environment, various collected soybean germplasm resources (including wild and cultivated soybean germplasm) are planted continuously for 2-3 years. Through repeated planting in multiple environments over many years, the influence of random factors such as climate fluctuations, soil microenvironment differences, and deviations in cultivation management details is effectively eliminated, ensuring the reliability and stability of the screening results and avoiding screening errors due to accidental performance in a single environment or year. From a breeding principle perspective, a uniform and stable selenium-rich environment allows the selenium absorption, translocation, and accumulation capabilities of different soybean germplasms to be fully expressed, avoiding misjudgments of traits caused by insufficient or excessive selenium supply, and allowing the true characteristics of each germplasm to be fully displayed. Based on this, on the one hand, individuals with a grain selenium content greater than 0.35 mg / kg were selected as high-selenium donors. These materials carry genetic material related to high selenium accumulation, which can provide a genetic basis for high selenium accumulation in subsequent hybrid offspring. On the other hand, individuals with a yield of not less than 150 kg / mu and a protein content of not less than 40% were selected as agronomically superior recipients. These materials have good production performance, which can ensure that the varieties bred later have both high selenium accumulation characteristics and the high-yield and high-quality traits required for actual production. Ultimately, a reasonable combination of high selenium accumulation traits and excellent agronomic traits is achieved, providing stable and high-quality parent materials for subsequent hybridization breeding and laying a solid foundation for the entire breeding process.

[0031] Step 2: Using the high-selenium donor material obtained in Step 1 as the male parent and the agronomically superior recipient material as the female parent, artificial hybridization was performed to obtain a first-generation population with highly consistent genotypes. This first-generation population was then self-crossed to obtain a second-generation genetically segregating population with widely segregating traits. Soybeans are self-pollinating crops, and the genotypes of all individuals in the first generation are basically identical, resulting in uniform phenotypic expression. However, after self-crossing of the first generation, genes controlling selenium accumulation, agronomical, and physiological traits undergo recombination and segregation, leading to significant differences in selenium absorption capacity, translocation efficiency, yield, and quality among individual plants in the second generation. This provides abundant variation material for subsequent indicator determination and threshold determination. In the second-generation population, two core physiological indicators were measured: root activity at flowering stage and the ratio of stem selenium content to grain selenium content at grain filling stage. Root activity directly reflects the soybean root system's ability to absorb selenium ions from the soil; higher activity indicates higher selenium content. The stronger the absorption efficiency, the higher the ratio of stem selenium content to grain selenium content, reflecting the efficiency of selenium transport and distribution from vegetative organs such as stems to grain sinks. The lower the ratio, the more fully selenium is transported to grains, and the higher the selenium accumulation in grains. Subsequently, based on the root activity value and stem-to-grain selenium content ratio corresponding to high-quality single plants with grain selenium content greater than 0.35 mg / kg, the thresholds for root activity at flowering period and stem-to-grain selenium content ratio at grain filling period were determined for subsequent generations. This transformed the subsequent screening from traditional visual phenotypic selection to quantitative index selection, significantly improving the accuracy, stability, and repeatability of breeding.

[0032] Step 3: Achieve efficient and precise selection of high-selenium single plants through a three-step progressive screening process; the first step is to conduct pre-screening based on the root vitality threshold during flowering and the selenium content ratio threshold between stems and grains during the grain-filling stage determined in Step 2. The flowering period is a crucial time for soybeans to achieve both vegetative and reproductive growth. At this stage, root vitality directly determines the plant's ability to continuously absorb selenium. The grain-filling stage is a critical phase for selenium accumulation in the grains, and the efficiency of selenium transport from the stem to the grains directly determines the final selenium content in the grains. Only individual plants that simultaneously meet the threshold values ​​of both indicators possess strong selenium absorption and grain transport capabilities, thus eliminating individuals with weak absorption and poor transport. The second step involves measuring the antioxidant enzyme activity of the individual plants selected in the first step, retaining those with antioxidant enzyme activity not lower than the set threshold. High concentrations of selenium can produce excessive reactive oxygen species in plants, causing oxidative stress and inhibiting normal plant growth and development. The antioxidant enzyme system can effectively scavenge reactive oxygen species and alleviate selenium toxicity. Therefore, the level of antioxidant enzyme activity in the seedling stage directly characterizes the soybean's tolerance to high-selenium environments; the higher the enzyme activity, the better the plant's growth stability under selenium-rich conditions. The third step involves further measuring the selenium content in the mature grains of the individual plants selected in the second step, screening out individual plants with a selenium content greater than 0.35 mg / kg, ultimately obtaining superior hybrid plants. The first and second steps are both indirect physiological indicator screenings, which can only reflect auxiliary characteristics related to selenium accumulation. Grain selenium content is the most direct and ultimate evaluation indicator of high selenium content. Through three-step screening, the comprehensive screening goal of "strong absorption, good translocation, high selenium tolerance, and grain selenium compliance" can be achieved.

[0033] Step 4: Continuously self-pollinate the superior hybrid plants obtained in Step 3 to obtain three, four, and five generations, strictly repeating the three-step screening process of Step 3 in each generation. Soybean is a self-pollinating crop. As the number of self-pollination generations increases, the genotype of the plant will continuously tend towards homozygosity. The gene combinations controlling desirable traits such as high selenium accumulation, high yield, and high quality can be stably inherited generation by generation. At the same time, repeating the same screening process in each generation can continuously eliminate low-selenium, low-yield, and inferior individual plants that appear due to trait segregation, ensuring that the target traits do not degenerate, and making the selected materials genetically stable and consistent with no trait segregation. After completing five generations of screening, the obtained stable lines are placed in different ecological regions for field identification. Because there are significant differences in soil selenium content, climate conditions, soil type, and cultivation conditions in different ecological regions, materials bred in a single environment may only be adapted to that specific environment. However, by simultaneously identifying multiple ecological regions, strains that are not sensitive to the environment and have wide adaptability can be screened out. This ensures that the selected soybean germplasm can maintain a stable selenium content of more than 0.35 mg / kg in grains when planted in various selenium-rich ecological regions, while maintaining excellent agronomic traits, thus making it truly valuable for large-scale application.

[0034] In one possible implementation, the drought resistance damage rate and cold resistance damage rate of the wild soybean germplasm described in step 1 are ≤30%.

[0035] Specifically, wild soybean germplasm is included in the soybean germplasm resources used in step 1, and the drought resistance damage rate is required to be no higher than 30% and the cold resistance damage rate is required to be no higher than 30%. Compared to conventionally cultivated soybeans, wild soybeans retain stronger resilience to adverse conditions, greater diversity of stress-resistant genes, and greater tolerance to abiotic stresses. Including wild soybeans as germplasm resources can introduce a superior genetic foundation for stress resistance in subsequent breeding of high-selenium soybeans. A drought resistance damage rate of ≤30% indicates that the wild soybean experiences less damage, such as inhibited growth, leaf damage, and reduced biomass, under drought stress, demonstrating stable drought adaptability. A cold resistance damage rate of ≤30% indicates that it can maintain normal physiological metabolism under low-temperature conditions and is less prone to cold damage, stunted growth, or death. By limiting these two stress resistance indicators, the selected parental materials can be guaranteed to not only possess the potential for high selenium accumulation, high yield, and high quality, but also outstanding drought and cold resistance. This will enable the subsequently bred soybean germplasm to better adapt to adverse natural environments such as drought and low temperatures when planted in selenium-enriched fields, improving plant survival rate, growth stability, and selenium accumulation stability under complex ecological conditions, ultimately achieving synergistic improvement of stress resistance, high selenium accumulation characteristics, and excellent agronomic traits.

[0036] This application achieves precise breeding and stable inheritance of high selenium-accumulating traits by long-term screening of parents in a stable selenium-rich environment, constructing hybrid segregating populations and determining key physiological index thresholds, combined with multi-generation progressive quantitative screening and multi-ecological region identification. This effectively improves the selenium accumulation capacity and trait stability of soybeans, while also taking into account excellent agronomic traits such as high yield and high protein. As a result, high selenium-accumulating soybean germplasm with wide adaptability and large-scale promotion has been obtained.

[0037] In one possible implementation, the root activity during flowering in step 2 is determined by the TTC method, and the selenium content in the stems and grains is determined by atomic fluorescence spectrometry.

[0038] Specifically, root activity during the flowering period was determined using the TTC method, or triphenyltetrazolium chloride method. The principle is that the TTC reagent can be reduced by dehydrogenases in the living cells of plant roots to produce red triphenylformazan. The stronger the root activity, the higher the dehydrogenase activity, and the more red substance is produced. Colorimetric determination quantitatively reflects the metabolic activity and absorption capacity of the roots. This method has good stability, high sensitivity, and strong repeatability, accurately reflecting the absorption capacity of soybean roots for soil nutrients and selenium during the flowering period, ensuring reliable screening results. Stem selenium content and grain selenium content were both determined using atomic fluorescence spectrometry, a national standard for selenium detection. This general method features low detection limits, high accuracy, minimal interference, and fast detection speed. It can accurately quantify the actual selenium content in soybean stems and seeds, thereby accurately calculating the ratio of stem selenium content to seed selenium content. This truly reflects the translocation and distribution patterns of selenium within soybean plants. By clearly defining the detection method, it ensures the consistency, accuracy, and comparability of detection results across different experiments, generations, and environments. This makes the root vigor threshold at flowering stage and the threshold for the ratio of stem selenium content to seed selenium content at grain filling stage determined in step 2 more scientific, objective, and repeatable, providing reliable data support for standardized screening in subsequent generations.

[0039] In one possible implementation, the antioxidant enzyme activity during the seedling stage in step 3 is superoxide dismutase activity.

[0040] Specifically, the main function of superoxide dismutase (SOD) is to efficiently remove toxic reactive oxygen species induced by the accumulation of high concentrations of selenium in plant cells, reduce oxidative damage, maintain cell membrane stability, and thus enhance the selenium tolerance and growth stability of soybeans in selenium-rich environments. The seedling stage is a critical period when soybean plants are most sensitive to environmental stress and rapidly establish their antioxidant system. Measuring SOD activity at this time can most accurately and sensitively reflect the selenium tolerance potential and stress resistance level of individual plants. Using it as a screening indicator can accurately eliminate individual plants that are sensitive to selenium stress and have weak growth, while retaining superior individual plants with strong selenium tolerance and stable physiological metabolism. This makes the screening results more targeted and scientific, and also provides a reliable stress resistance guarantee for the stable growth of high-selenium soybean germplasm in different selenium-rich ecological regions, ensuring that the selected germplasm has both high selenium accumulation characteristics and excellent stress resistance and environmental adaptability.

[0041] In one possible implementation, the antioxidant enzyme activity described in step 3 is determined using the nitroblue tetrazolium method.

[0042] Specifically, the nitroblue tetrazolium method is a commonly used standard method in plant physiology for determining superoxide dismutase (SOD) activity. Its principle is that nitroblue tetrazolium undergoes a photoreduction reaction under light conditions, which SOD inhibits. The higher the enzyme activity, the more significant the inhibitory effect. By detecting changes in the absorbance of the reaction system, the activity level of SOD can be quantitatively calculated. This method accurately reflects the antioxidant capacity and stress tolerance of soybean seedlings in a selenium-rich environment. Furthermore, the method exhibits good stability and high repeatability, ensuring good consistency and comparability of enzyme activity measurements among different individual plants and generations. This makes the screening process more standardized and reliable, further improving the accuracy and stability of high-selenium soybean germplasm screening.

[0043] In one possible implementation, during the determination using the nitroblue tetrazolium method, the reaction temperature is controlled at 20-30°C and the reaction time is controlled at 10-20 min.

[0044] Specifically, in the process of determining superoxide dismutase (SOD) activity in soybean seedlings using the nitroblue tetrazolium method, the reaction temperature is controlled at 20-30℃ and the reaction time at 10-20 min. This temperature range matches the physiological metabolic temperature of soybean plants, ensuring that the enzymatic and photoreduction reactions are in a stable, mild, and suitable state. This avoids instability caused by excessively high temperatures and low reaction rates caused by excessively low temperatures, thus ensuring the stability and accuracy of the detection results. Simultaneously, controlling the reaction time within a reasonable range ensures that the photoreduction reaction of nitroblue tetrazolium proceeds fully, allowing the inhibitory effect of SOD to be fully realized, while avoiding deviations caused by excessively long reaction times and inaccurate numerical measurements caused by excessively short reaction times. By uniformly and precisely controlling the key reaction conditions, the interference of external factors on the enzyme activity measurement results can be effectively reduced, improving the consistency and comparability of detection data from different samples and batches. This makes the screening criteria more unified and objective, further enhancing the standardization and reliability of the high-selenium soybean germplasm screening process.

[0045] In one possible implementation, step 3, which involves pre-screening individual plants based on the root vigor threshold during flowering and the ratio threshold of stem selenium content to grain selenium content during grain filling, specifically includes:

[0046] A dual-weighted scoring method was used to evaluate root activity during flowering and the ratio of stem to grain selenium content during grain filling. The weighting coefficient for the ratio of stem to grain selenium content during grain filling was 0.7, and the weighting coefficient for root activity during flowering was 0.3. Individual plants with root activity during flowering not lower than the threshold value and a ratio of stem to grain selenium content during grain filling lower than the threshold value, and whose overall score reached the preset threshold value, were retained.

[0047] In one possible implementation, in step 4, the field identification adopts simultaneous planting in multiple selenium-rich gradient areas. The different ecological areas include at least a high-selenium area with soil selenium content of 0.4-0.5 mg / kg and a medium-high selenium area with soil selenium content of 0.2-0.4 mg / kg, and the planting density in each ecological area is 10,000-15,000 plants / acre.

[0048] Specifically, in step 4, when conducting field identification of the strains obtained from five generations of screening, a method of simultaneous planting in multiple selenium-enriched gradient areas was adopted. This method clearly identifies at least two typical selenium-enriched ecological regions: high-selenium areas with soil selenium content of 0.4-0.5 mg / kg and medium-high selenium areas with soil selenium content of 0.2-0.4 mg / kg. Simultaneously, the planting density in each ecological region was uniformly limited to 10,000-15,000 plants / acre. By setting up identification areas with different soil selenium content gradients, the selenium accumulation stability, growth adaptability, and agronomical characteristics of the target strains under different selenium-enriched environments can be comprehensively examined. This approach avoids the biased results caused by identification in a single selenium-rich region, ensuring that the selected soybean germplasm can stably achieve the required selenium content in grains under various selenium-rich ecological conditions, including medium-high selenium and high selenium. Unifying the planting density in different ecological regions can eliminate the interference of cultivation factors such as population structure, nutrient competition, and field ventilation and light conditions on the identification results, ensuring good comparability and objectivity of the phenotypic performance of the lines in different regions. This makes the field identification results more scientific, accurate, and reliable, and ultimately selects high-selenium soybean germplasm with stable selenium accumulation traits, wide environmental adaptability, and suitability for large-scale promotion and planting.

[0049] In one possible implementation, during the screening process in steps 3 and 4, individual plants with a selenium content greater than 0.35 mg / kg but a protein content less than 40% are removed.

[0050] Specifically, in step 3, the screening of the second-generation genetic segregating population, and in step 4, the continuous screening of the third, fourth, and fifth-generation lines, a simultaneous screening step for protein content was added. Individual plants with a selenium content greater than 0.35 mg / kg but a protein content less than 40% were specifically removed. This operation can ensure the stable inheritance of soybean nutritional quality traits. During the self-pollination process of soybean hybrids, the trait controlling selenium accumulation and the trait controlling protein content can independently segregate and recombine. Even if agronomically superior materials with a protein content of not less than 40% are selected as parents in step 1, subsequent generations will still produce segregating plants with adequate selenium content but decreased protein content, failing to meet the nutritional standards and production application requirements of high-quality soybeans. By continuously eliminating plants with adequate selenium content but insufficient protein content in multiple generations of screening, superior plants with both high selenium accumulation and high protein nutritional quality can be retained, effectively avoiding the loss of superior traits caused by trait segregation. This ensures that the soybean germplasm obtained by the final breeding meets the core objective of high selenium accumulation while maintaining a stable protein content of not less than 40%, achieving the synergistic and stable inheritance of the high selenium accumulation trait and the superior agronomic trait of high protein. This further enhances the rigor and completeness of the breeding method, as well as the practicality and commercial value of the bred germplasm.

[0051] In one possible implementation, the selenium content of the grains is detected when the grains are fully mature and naturally air-dried to a moisture content of 12-14%.

[0052] Specifically, testing is conducted under the condition that the seeds are fully mature and naturally air-dried to a moisture content of 12–14%. Fully mature seeds are a prerequisite for ensuring that the accumulation of selenium in soybeans reaches its final stable state. This avoids the possibility of low selenium content readings due to incomplete seed development. Naturally air-drying the seeds to a moisture content of 12–14% is not only the standard moisture content condition for soybean seed storage and quality testing, but also a key control condition to ensure the comparability of test results between samples from different individual plants, batches, and years. This effectively eliminates the deviation in selenium content calculation caused by differences in moisture content, ensuring that the test results are true, stable, and accurate. This provides reliable data for the screening of high-selenium soybeans across generations, further improving the standardization and consistency of the entire breeding process.

[0053] The present application is further illustrated by the following embodiments.

[0054] 1. Test materials and test environment

[0055] The experimental materials used in this embodiment consisted of 46 soybean germplasm resources that had been collected, sorted, and identified for drought resistance and cold tolerance. These included 8 wild soybean germplasm resources and 38 cultivated soybean germplasm resources. All germplasm resources were previously planted continuously for 2-3 years in selenium-rich areas with soil selenium content of 0.4–0.5 mg / kg to complete basic identification of grain selenium accumulation capacity, yield, protein content, and agronomic traits. All wild soybean germplasm resources showed drought resistance and cold tolerance damage rates of ≤30%, indicating good overall stress resistance. Significant differences existed in selenium accumulation capacity, yield, and quality traits among different germplasm resources, allowing for the screening of high-selenium donor materials and agronomically superior recipient materials. The experiment was conducted in a selenium-rich area where the soil selenium content remained stable at 0.43–0.48 mg / kg. The soil type was medium loam, with an organic matter content of 15.6–18.3 g / kg and a pH of 6.8–7.2. The previous crop was maize. The plots were flat and had uniform fertility. All experimental materials were subject to uniform field management measures, including sowing time, fertilization amount, irrigation method, and pest and disease control, to ensure that the experimental environment was consistent with the cultivation conditions and to eliminate interference from non-experimental factors on phenotypic performance.

[0056] 2. Screening of parental materials: In selenium-enriched experimental fields with soil selenium content of 0.43–0.48 mg / kg, 46 soybean germplasm resources were screened in the field for three consecutive years. Sowing was done manually in early May each year, with a row spacing of 40 cm and a plant spacing of 10 cm. After maturity, the seeds were harvested manually and air-dried naturally until the moisture content reached 12%–14% before various indicators were tested. Selenium content was determined by atomic fluorescence spectrometry, protein content by the Kjeldahl method, and yield was converted to per-acre yield after plot measurements. The results of the three consecutive years of screening showed significant differences in selenium accumulation, yield, and protein content among different germplasms. Finally, three high-selenium donor materials and five agronomically superior recipient materials were screened. The screening results are shown in Table 1.

[0057] Table 1 Results of parental material screening in Step 1

[0058] High selenium donor W-04 0.46 142 39.2 High selenium donor W-06 0.42 145 38.7 High selenium donor W-08 0.39 140 39.5 Excellent receptors C-12 0.26 182 43.2 Excellent receptors C-18 0.24 175 42.6 Excellent receptors C-25 0.23 168 41.8 Excellent receptors C-31 0.25 162 41.2 Excellent receptors C-36 0.22 156 40.5

[0059] Ultimately, the wild soybean germplasm W-04, which has the highest selenium content in its grains, was selected as the male parent, and the cultivated soybean germplasm C-12, which has the best yield and protein traits, was selected as the female parent for subsequent hybridization.

[0060] 3. Construction of hybrid populations and determination of indicator thresholds

[0061] Artificial hybridization was performed using the high-selenium donor W-04 as the male parent and the agronomically superior recipient C-12 as the female parent to obtain first-generation plants with identical genotypes. These first-generation plants were self-pollinated to produce seeds, resulting in a second-generation genetic segregating population of 328 plants with widely segregating traits. Core physiological indicators and selenium content were measured in the second-generation population. Root vigor at flowering time was determined using the TTC method. Specifically, 0.5g of fresh white roots were added to 5mL each of 0.4% TTC solution and phosphate buffer, and the mixture was reacted at 37℃ in the dark for 2 hours. The reaction was terminated by adding 1mol / L sulfuric acid, and the red extract was obtained by thorough grinding with ethyl acetate. The product was diluted to 10 mL, and the absorbance was measured at a wavelength of 485 nm to calculate root activity. Stem samples were collected during the grain-filling stage, and grain samples were collected during the maturity stage. The samples were blanched at 105℃ for 30 min, dried at 70℃ to constant weight, pulverized through a 100-mesh sieve, and 0.2 g of the sample was weighed and digested with a nitric acid-perchloric acid mixture until clear. The volume was then adjusted, and the selenium content of the stems and grains was determined by atomic fluorescence spectrometry, and the ratio between the two was calculated. Based on the data of superior single plants with a grain selenium content > 0.35 mg / kg, the standardized screening threshold was determined. The measurement range and threshold of each index are shown in Table 2.

[0062] Table 2. Results of Second-Generation Indicator Measurement and Screening Thresholds

[0063] Root activity during flowering (U / g・h) 22.4-41.8 ≥32.6 Stem selenium content / grain selenium content 0.62-1.18 ≤0.85 Selenium content in grains (mg / kg) 0.21-0.49 ＞0.35 Superoxide dismutase (SOD) activity during seedling stage (U / g·min) 68.5-132.6 ≥95.0

[0064] 4. Second-generation three-step screening

[0065] A progressive three-step screening process was conducted on 328 second-generation plants. The first step involved a dual-weighted scoring system based on the root vitality threshold at flowering stage and the stem-to-seed selenium content ratio threshold. The stem-to-seed selenium content ratio at the grain-filling stage had a weight of 0.7, and root vitality at flowering stage had a weight of 0.3. 89 individual plants that simultaneously met both criteria and achieved a comprehensive weighted score reaching the threshold were retained. The weighted score was calculated as follows: root vitality at flowering stage and stem-to-seed selenium ratio at grain-filling stage were normalized to a score of 0-100. The comprehensive score was calculated as: root vitality normalized score × 0.3 + stem-to-seed selenium ratio normalized score at grain-filling stage × 0.7. The preset comprehensive score threshold was 80 points. The second step involved determining the superoxide dismutase (SOD) activity of the retained plants at the seedling stage using the nitroblue tetrazolium method, taking 0.5g of fresh leaf samples. The mixture was ground in a pre-cooled phosphate buffer solution in an ice bath and centrifuged at 12000 r / min for 20 min. The supernatant was used as the enzyme solution. The reaction system contained phosphate buffer solution, methionine, nitroblue tetrazolium, EDTA, enzyme solution, and riboflavin. The reaction was carried out under 4000 lx light and 25℃ for 15 min. After the reaction was terminated in the dark, the absorbance was measured at 560 nm and the enzyme activity was calculated. 42 plants with enzyme activity not lower than the threshold were retained. In the third step, the selenium content and protein content of the grains of the plants retained in the second step were measured at maturity. The selenium content of the grains was measured after the grains were fully mature and naturally air-dried to a moisture content of 12% to 14%. At the same time, plants with a selenium content >0.35 mg / kg but a protein content <40% were strictly removed. Finally, 16 superior hybrid plants were obtained. The specific screening results are shown in Table 3.

[0066] Table 3. Statistics on the number of second-generation three-step screenings

[0067] Step 1: Weighted scoring screening Root activity ≥ root activity threshold at flowering stage, ratio of stem selenium content to grain selenium content at grain filling stage ≤ threshold, and overall score ≥ overall score threshold. 89 Step 2: Screening for antioxidant enzyme activity Superoxide dismutase activity ≥ set threshold 42 Step 3: Selenium content and protein screening Selenium > 0.35 mg / kg and protein ≥ 40% 16

[0068] 5. Multi-generational continuous self-pollination purification and field identification in multiple ecoregions

[0069] The 16 hybrid superior plants obtained in step 3 were continuously self-pollinated to obtain third, fourth, and fifth generation plants. The three-step screening process of step 3 was strictly repeated in each generation. At the same time, individual plants with selenium content meeting the standard but protein content below 40% due to phenotypic segregation were continuously removed. Individuals with low yield, low selenium, and poor stress resistance were eliminated generation by generation to continuously homozygous and stabilize the excellent traits of high selenium accumulation, high yield, high protein, and strong stress resistance. A total of 6 genetically stable and uniform superior lines were obtained after five generations. Field identification of the 6 stable lines was carried out in multiple selenium-enriched gradient ecological areas. The identification areas included at least a high selenium area with soil selenium content of 0.42-0.49 mg / kg and a medium-high selenium area with soil selenium content of 0.23-0.38 mg / kg. The planting density in both ecological areas was uniformly 13,000 plants / mu. A randomized block design was used with 3 replicates. The plants were managed uniformly throughout the growth period. After maturity, the selenium content, protein content, and yield of the grains were measured. The identification results are shown in Table 4.

[0070] Table 4. Results of multi-ecoregional identification of five generations of stable strains

[0071] S-01 0.47 0.40 42.8 178 S-02 0.44 0.38 42.1 172 S-03 0.42 0.37 41.6 168 S-04 0.40 0.36 41.2 163 S-05 0.39 0.36 40.8 158 S-06 0.36 0.35 40.3 152

[0072] Through the breeding process described in this embodiment, including parental selection, hybrid population construction, determination of index thresholds, three-step weighted selection, multi-generation self-pollination purification, and multi-ecological zone identification, six genetically stable, high-selenium soybean lines with excellent traits were finally obtained. When planted in high-selenium and medium-high-selenium areas, all lines had a selenium content greater than 0.35 mg / kg, a protein content of no less than 40%, and a yield of ≥150 kg / mu, demonstrating excellent overall performance. Among them, lines S-01 and S-02 showed the most outstanding performance in selenium accumulation capacity, yield, protein content, and environmental adaptability, and can be used as high-selenium soybean germplasm that meet the requirements for production application, and can be demonstrated and promoted in medium-high-selenium and high-selenium ecological zones.

[0073] 6. Verification test of selected strains

[0074] 6.1 Validation Materials

[0075] The six stable fifth-generation high-selenium soybean lines (S-01, S-02, S-03, S-04, S-05, and S-06) obtained in this application were used as verification materials. All the seeds of these lines were pure seeds that were fully mature, naturally air-dried to a moisture content of 12%–14%, with a purity of ≥99% and a germination rate of ≥85%. Locally cultivated conventional soybean varieties were set as control materials for the verification experiment. All verification samples were uniformly numbered, stored, and managed.

[0076] 6.2 Verification Experiment Design

[0077] The validation experiment was conducted simultaneously at two ecological test sites: a high-selenium area (soil selenium content 0.42–0.49 mg / kg) and a medium-high selenium area (soil selenium content 0.23–0.38 mg / kg). The experiment adopted a randomized block design with three biological replicates. The plot area was 10 m², the row length was 5 m, the row spacing was 40 cm, the plant spacing was 10 cm, and the planting density was uniformly 12,000 plants / mu. All experimental materials adopted the same sowing time (early May), the same fertilizer formula (15 kg / mu of diammonium phosphate as base fertilizer and 5 kg / mu of urea as top dressing), the same irrigation system, the same weeding method, and the same green pest control measures. The validation period was two consecutive natural production years, and two consecutive generations of self-pollination (sixth and seventh generations). All sample collection, index testing, and data recording were carried out by designated personnel according to unified standards. Blank controls and standard sample calibration were set up throughout the process to ensure that the validation process was scientific and standardized and the validation results were accurate and reproducible.

[0078] 6.3 Verification of intergenerational genetic stability

[0079] 6.3.1 Verification Objective

[0080] The study aimed to verify whether the selenium content, protein content, yield, and agronomic traits of the six strains could be stably inherited during the fifth, sixth, and seventh generations of continuous self-pollination, and whether phenomena such as trait segregation, genetic variation, and trait degeneration occurred, in order to confirm whether the strains had reached a genetically homozygous stable state.

[0081] 6.3.2 Verification Operation Procedure

[0082] 1. Six fifth-generation line seeds were sown according to a unified experimental design, and conventional field management was carried out until maturity. Individual plants were harvested, threshed, and dried to obtain sixth-generation seeds.

[0083] 2. Continue to sow six generations using the same field design and management process, and harvest them all at once after they mature to obtain seven generations of seeds;

[0084] 3. The fifth, sixth, and seventh generations were all planted in the same ecological zone, under the same soil conditions, and under the same management model to ensure that the experimental conditions were completely consistent across generations;

[0085] 4. After each generation matures, three replicate plots are randomly selected from each line, and ten plants are randomly selected from each plot for mixed sampling. The grains are then naturally air-dried to a moisture content of 12%–14%.

[0086] 5. The selenium content of the grains was determined by atomic fluorescence spectrometry, and the protein content was determined by the Kjeldahl method. The yield per mu was calculated after actual harvesting in each plot.

[0087] 6. Observe and record the plant growth, plant height, number of branches on the main stem, flowering period, maturity period, disease resistance, and lodging resistance in the field throughout the entire growth period to determine whether phenotypic segregation has occurred.

[0088] 6.3.3 Verification Results

[0089] As shown in Table 5.

[0090] Table 5 Results of intergenerational genetic stability verification

[0091] S-01 Five Dynasties 0.47 42.8 178 No separation, no variation Stablize S-01 Sixth generation 0.46 42.7 177 No separation, no variation Stablize S-01 Seven generations 0.47 42.8 178 No separation, no variation Stablize S-02 Five Dynasties 0.44 42.1 172 No separation, no variation Stablize S-02 Sixth generation 0.43 42.0 171 No separation, no variation Stablize S-02 Seven generations 0.44 42.1 172 No separation, no variation Stablize S-03 Five Dynasties 0.42 41.6 168 No separation, no variation Stablize S-03 Sixth generation 0.41 41.5 167 No separation, no variation Stablize S-03 Seven generations 0.42 41.6 168 No separation, no variation Stablize S-04 Five Dynasties - Seven Dynasties 0.39-0.40 41.1-41.2 162-163 No separation, no variation Stablize S-05 Five Dynasties - Seven Dynasties 0.38-0.39 40.7-40.8 157-158 No separation, no variation Stablize S-06 Five Dynasties - Seven Dynasties 0.35-0.36 40.2-40.3 151-152 No separation, no variation Stablize

[0092] 6.3.4 Verification Conclusion

[0093] The six strains showed stable expression of selenium content, protein content, and yield in grains in different years and soils with different selenium contents, with no fluctuations of less than 5%. They also showed strong ecological adaptability and were suitable for large-scale promotion.

[0094] 6.4 Verification of uniformity of agronomic traits

[0095] 6.4.1 Verification Objective

[0096] Verify the uniformity and consistency of phenotypes among individual plants within the line to meet the requirements of specificity, uniformity, and stability (DUS) of superior germplasm.

[0097] 6.4.2 Verification Operation Procedure

[0098] 1. Select representative plots of each strain that are uniformly growing and free from disease;

[0099] 2. Randomly select 100 individual plants from each strain, label them, and conduct a fixed-point survey throughout the entire growth period;

[0100] 3. Investigate traits: plant height, number of branches on the main stem, flowering period, maturity period, field disease resistance, and lodging resistance;

[0101] 4. Calculate the mean, standard deviation, and coefficient of variation (CV) for each trait. Criterion: CV < 5% indicates uniformity.

[0102] 6.4.3 Verification Results

[0103] As shown in Table 6.

[0104] Table 6. Verification results of uniformity in agronomic traits

[0105] S-01 3.12 2.86 ≤1 Neat and uniform S-02 3.25 2.91 ≤1 Neat and uniform S-03 3.01 2.77 ≤1 Neat and uniform S-04 2.94 2.69 ≤1 Neat and uniform S-05 3.36 2.98 ≤1 Neat and uniform S-06 3.42 3.05 ≤1 Neat and uniform

[0106] 6.4.4 Verification Conclusion

[0107] The coefficients of variation for all agronomic traits of the six strains were all <5%, with minimal differences between plants. The plants exhibited uniform growth, maturity, and resistance, meeting the standards for consistency of superior germplasm.

[0108] 6.5 Repeatability Validation of the Detection Method

[0109] 6.5.1 Verification Objective

[0110] Verify the precision, accuracy, and repeatability of the methods for detecting selenium and protein content in grains to ensure that the test data are authentic, reliable, repeatable, and traceable.

[0111] 6.5.2 Verification Operation Procedure

[0112] 1. Take three parallel and independent samples from each line of mixed grain samples, and number them as Parallel Sample 1, Parallel Sample 2, and Parallel Sample 3;

[0113] 2. Each of the three samples was independently pulverized, weighed, digested, diluted to volume, and tested using the instrument.

[0114] 3. Selenium content was determined independently three times using atomic fluorescence spectrometry, and protein content was determined independently three times using the Kjeldahl method.

[0115] 4. Calculate the relative standard deviation (RSD) of the three parallel test results. The judgment criterion is: RSD < 5% is considered as the test method being qualified.

[0116] 6.5.3 Verification Results

[0117] As shown in Table 7.

[0118] Table 7 Results of Repeatability Validation of Detection Methods

[0119] S-01 1.22 0.95 qualified S-02 1.45 1.01 qualified S-03 1.68 1.06 qualified S-04 1.93 1.12 qualified S-05 2.41 1.18 qualified S-06 3.08 1.26 qualified

[0120] 6.5.4 Verification Conclusion

[0121] All test results had an RSD well below 5%, indicating that the test method had high precision and good repeatability, and the test data were true, accurate, and reliable.

[0122] 6.6 Comprehensive Verification Conclusion

[0123] Systematic, comprehensive, and standardized verification through five aspects—intergenerational genetic stability, annual ecological stability, uniformity of agronomic traits, core quality compliance, and repeatability of testing methods—shows that the six high-selenium soybean lines bred in this application are genetically stable, exhibit no phenotypic segregation, possess strong ecological adaptability, consistently meet quality standards, and provide accurate and reliable testing data. All lines have a selenium content >0.35 mg / kg, a protein content ≥40%, and a yield ≥150 kg / mu, fully meeting the breeding goals of high selenium accumulation, high protein, and high yield. Among them, lines S-01 and S-02 demonstrate the most outstanding performance in selenium accumulation capacity, protein content, yield level, environmental adaptability, and phenotypic uniformity, exhibiting the best overall performance. They can be used as stable and excellent high-selenium soybean germplasm for large-scale demonstration, promotion, and production application in medium-high selenium and high-selenium ecological zones.

[0124] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for breeding high-selenium soybean germplasm, characterized in that, include: Step 1: Plant soybean germplasm resources in selenium-rich areas with soil selenium content of 0.4-0.5 mg / kg for 2-3 years. The sources of soybean germplasm resources include wild soybean germplasm and cultivated soybean germplasm. Screen high selenium donor materials with grain selenium content greater than 0.35 mg / kg and agronomically excellent recipient materials with a yield greater than or equal to 150 kg / mu and a protein content greater than or equal to 40%. Step 2: Using the high-selenium donor material as the male parent and the agronomically superior recipient material as the female parent, hybridize to obtain the first generation, and self-pollinate to obtain the second generation genetic segregating population. Measure the ratio of root activity at flowering time to stem selenium content and grain selenium content at the grain-filling stage in the second generation genetic segregating population. Based on the ratio of root activity at flowering time to stem selenium content and grain selenium content at the grain-filling stage corresponding to individual plants with grain selenium content greater than 0.35 mg / kg, determine the threshold for root activity at flowering time and the threshold for the ratio of stem selenium content and grain selenium content at the grain-filling stage. Step 3: Perform a three-step screening on the second-generation genetic segregation population: First, pre-screen individual plants based on the root vigor threshold at flowering time and the stem-to-seed selenium content ratio threshold at the grain-filling stage; Second, measure the antioxidant enzyme activity of the pre-screened individual plants at the seedling stage, and retain individual plants with antioxidant enzyme activity not lower than the activity threshold; Third, from the individual plants retained in the second step, screen for individual plants with a mature grain selenium content greater than 0.35 mg / kg to obtain superior hybrid plants; Step 4: Self-pollinate the hybrid superior plants to obtain three generations, and perform the three-step screening in Step 3 on the three generations; self-pollinate the single plants obtained from the three generations to obtain four generations, and perform the three-step screening in Step 3 on the four generations; then self-pollinate the single plants obtained from the four generations to obtain five generations, and perform the three-step screening in Step 3 on the five generations; conduct field identification of the lines obtained from the five generations in different ecological regions, and select soybean germplasm with a grain selenium content greater than 0.35 mg / kg when planted in different ecological regions.

2. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, The drought resistance damage rate and cold resistance damage rate of the wild soybean germplasm mentioned in step 1 are ≤30%.

3. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, In step 2, the root activity during the flowering period was determined using the TTC method, and the selenium content in the stems and grains was determined using atomic fluorescence spectrometry.

4. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, The antioxidant enzyme activity in step 3 during the seedling stage is superoxide dismutase activity.

5. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, The antioxidant enzyme activity described in step 3 was determined using the nitroblue tetrazolium method.

6. The method for breeding high-selenium soybean germplasm according to claim 5, characterized in that, During the determination using the nitroblue tetrazolium method, the reaction temperature is controlled at 20-30℃ and the reaction time is controlled at 10-20 min.

7. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, Step 3, which involves pre-screening individual plants based on the root vitality threshold during flowering and the ratio threshold of stem selenium content to grain selenium content during grain filling, specifically includes: A dual-weighted scoring method was used to evaluate root activity during flowering and the ratio of stem to grain selenium content during grain filling. The weighting coefficient for the ratio of stem to grain selenium content during grain filling was 0.7, and the weighting coefficient for root activity during flowering was 0.

3. Individual plants with root activity during flowering not lower than the threshold value and a ratio of stem to grain selenium content during grain filling lower than the threshold value, and whose overall score reached the preset threshold value, were retained.

8. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, In step 4, the field identification adopts simultaneous planting in multiple selenium-rich gradient areas. The different ecological areas include at least a high selenium area with soil selenium content of 0.4-0.5 mg / kg and a medium-high selenium area with soil selenium content of 0.2-0.4 mg / kg, and the planting density in each ecological area is 10,000-15,200 plants / mu.

9. The method for breeding high-selenium soybean germplasm according to claim 8, characterized in that, During the screening process in steps 3 and 4, individual plants with a selenium content greater than 0.35 mg / kg but a protein content less than 40% are removed.

10. The method for breeding high-selenium soybean germplasm according to claim 1, characterized in that, The selenium content of the grains was detected when the grains were fully mature and naturally air-dried to a moisture content of 12-14%.

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