Multi-technology integrated rapid breeding method for soybean and application

Abstract

The application discloses a soybean rapid directional backcross transformation method and application, and belongs to the technical field of soybean breeding. The method takes 315 soybean cultivars covering different ecological zones as receptor parents, and takes glyphosate-resistant soybean Zhonghuang 6106 as a donor parent to perform soybean directional backcross transformation. In the hybridization stage, light, temperature, water and fertilizer, and hybridization technology are improved in four directions, so that the hybridization success rate is maximally improved, a large number of usable offspring are provided for the subsequent backcross process, and screening is performed on hybrid offspring and backcross offspring. The screening method is improved, phenotype screening, specific molecular marker screening and background recovery rate screening are combined, the screening efficiency is significantly improved, the workload is reduced, the breeding time is maximally shortened, and the breeding efficiency is improved. The whole backcross transformation process can be completed within 2 years, compared with 6 years of the traditional method, and significant progress is achieved.

Description

Technical Field

[0001] This invention relates to the field of soybean breeding technology, and more specifically to a rapid soybean breeding method and its application that integrates multiple technologies. Background Technology

[0002] Soybean backcross breeding is a plant breeding strategy primarily used to transfer a specific desirable trait from a donor parent to the genetic background of a recipient (recurrent) parent. This method is suitable for situations where it is desirable to retain all desirable traits of the recipient parent while introducing only a specific trait from the donor parent. It falls under the category of hybridization breeding. Backcross breeding includes the following basic steps: Initial hybridization: First, a donor parent with the target trait (e.g., glyphosate tolerance) is selected and crossed with a recipient (recurrent) parent with desirable agronomic traits. Continuous backcrossing: Next, the F1 generation is crossed again with the recipient parent (i.e., the first backcross), and then individuals carrying the target trait are selected from the offspring. This process can be repeated multiple times to ensure that the final variety retains as much of the recipient parent's genetic background as possible. Self-pollination homozygosity: When a series of backcrosses have sufficiently approximated the recipient parent's background, the final step is to self-pollinate the selected carriers of the target trait to fix the trait and obtain a new line with a relatively homozygous genotype. In the backcross breeding process, based on considerations of genetic diversity, a large number of recipient parent materials from different ecological regions are used to cross and backcross with donor parents. The above operations help to discover better trait combinations.

[0003] Generally, the backcross breeding cycle for a new variety is 8-10 years. Even with southern breeding and generation extension, it still takes at least 4-5 years to complete an experimental cycle. The cycle is long and the breeding efficiency is low. Moreover, the backcross breeding process involves not only the hybridization process of soybeans but also the backcross process. The floral organs of soybeans consist of bracts, 5 sepals, and 5 separate petals (1 large standard petal at the tip, 2 wing petals, and 2 keel petals) tightly surrounding the stamen and pistil. It is a typical self-pollinating crop. Studies have shown that due to the special structure of the petals, soybean hybridization is difficult, and the cross-pollination rate is very low, only 0.03-2.43%.

[0004] When soybeans from different ecological zones are simultaneously planted and hybridized in Hainan, the short sunshine hours in Hainan (approximately 14 hours) and the minimum temperature below 20℃ from mid-December to early February of the following year affect the hybridization of light and temperature-sensitive crops like soybeans. This results in small flower organs, short flowering time, and soybeans being unable to strictly close their flowers for pollination under short-day conditions, making artificial hybridization pollination impossible. Additionally, there is the problem of soybeans flowering at different times in different ecological zones.

[0005] When soybeans from different ecological zones are planted simultaneously in Beijing, the city's long-day climate prevents southern soybeans from maturing properly, increasing the difficulty of coinciding their flowering periods. Furthermore, the hybridization period for Beijing soybeans spans three months, from July to October, with hot and rainy summers occurring concurrently. This means that most of the soybean's growth and flowering stages coincide with the rainy season, and the humid climate is unfavorable for pollen dispersal. These factors severely limit the soybean hybridization process, resulting in low hybridization efficiency and low survival rates.

[0006] In the backcrossing process, dozens or even more backcross progeny need to be selected from each backcross generation and backcrossed with the recurrent parents. This backcrossing process needs to be repeated multiple times to achieve a background recovery rate at the standard level. However, in practice, as mentioned above, due to the unique flower structure and biological characteristics of soybeans, hybridization is inherently challenging. The enormous workload of backcrossing each generation undoubtedly places immense pressure and resource consumption on research, severely restricting the improvement of breeding efficiency.

[0007] Therefore, how to provide a rapid and targeted backcrossing method for soybeans to improve the efficiency of targeted backcrossing is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a multi-technology integrated rapid soybean breeding method and its application, which significantly improves the efficiency of directional backcrossing and conversion. Using the method of the present invention, 315 soybean varieties (lines) can be rapidly backcrossed and converted with Zhonghuang 6106 within 2 years to obtain new germplasm. Compared with the traditional method of 6 years, the conversion process is greatly shortened and the breeding efficiency is improved.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A rapid directional backcross breeding method for soybeans includes the following steps:

[0011] (1) Selection of donor and recipient parents: Soybean varieties with glyphosate tolerance were selected as donor parents, and soybean varieties with excellent traits from the southern region, the Huang-Huai region, the northeast region and / or the northwest region were selected as recipient parents;

[0012] (2) Hybridization: The donor parent and the recipient parent are hybridized, and hybrid grains are harvested;

[0013] (3) F1 generation screening: The hybrid seeds harvested in step (2) are sown, and resistance screening, agronomic trait screening, and background detection are carried out in sequence using KASP molecular markers, agronomic trait phenotypes, and Zhongdouxin No. 1 chip. Plants with the same or similar agronomic traits as the recipient parent, high background recovery rate, high similarity to the recurrent parent, and glyphosate tolerance are selected for use.

[0014] (4) Backcrossing: The plants selected in step (3) are backcrossed with the recipient parent, and BC1F0 is harvested;

[0015] (5) BC1F1 generation screening: Sow the BC1F0 harvested in step (4), and combine KASP molecular markers, agronomic phenotypic and Zhongdouxin No. 1 chip to carry out resistance screening, agronomic phenotypic screening and background detection in sequence. In BC1F1, select 5-7 plants with agronomic traits that are the same or similar to the recipient parent, high background recovery rate, high similarity to the recurrent parent and glyphosate tolerance, and keep them for later use.

[0016] (6) Repeated backcrossing: The plants obtained in step (5) are backcrossed with the recipient parent multiple times. The screening process of BC1F1 generation is repeated for each backcross generation. 5-7 plants from the backcross generation are selected for the next backcross. Through multiple backcrossings, when the SNP detection similarity rate between the parent and offspring of Zhongdouxin No. 1 is >97%, the SSR marker detection is carried out to meet the requirements of variety approval. Plants that meet the requirements of the number of differences in backcross SSR markers in the "National Transgenic Soybean Variety Approval Standard (Trial)" and also have glyphosate tolerance are obtained.

[0017] (7) Self-pollination homozygosity: The plants obtained in step (6) are self-pollinated homozygosity. The self-pollinated offspring are subjected to photoperiod regulation of 11h light / 13h dark from the time the true leaves of soybean unfold. When the plants enter the grain-filling stage, natural light and longitude management can be carried out to accelerate generation homozygosity and obtain new germplasm.

[0018] Preferably, the hybridization method in step (2) is as follows:

[0019] When the hybridization location is Beijing, the specific hybridization method is as follows:

[0020] A: Sowing: Sowing the donor parent and the recipient parent from different ecological zones in the Beijing area;

[0021] B: Photoperiod Adjustment: Photoperiod adjustment was performed on parental varieties from different ecological zones. The photoperiod was adjusted to 8 hours of light / 16 hours of darkness for soybean varieties in southern regions; 12 hours of light / 12 hours of darkness for soybean varieties in the Huang-Huai region; 16 hours of light / 8 hours of darkness for soybean varieties in Northeast China; and 16 hours of light / 8 hours of darkness for soybean varieties in Northwest China. This shortened and homogenized the flowering period while extending the flowering duration. The flowering period refers to the time from seedling emergence to initial flowering; the flowering duration refers to the time from initial flowering to pod formation.

[0022] C: Temperature compensation measures: After soybeans are sown in April or May each year, mulching is carried out. When the outdoor temperature is ≥20℃, mulching is stopped.

[0023] D: Integrated water and fertilizer management: Step A: Apply slow-release compound fertilizer as base fertilizer to the soil at the same time as soybean sowing. From the V3 stage of soybean, apply urea, amino acids, trace elements and growth regulators as top dressing; routine water management.

[0024] E: Hybridization technique: Males are removed and pollinated on the same day. Leaves are immediately wrapped after pollination. Hybridization is complete. The wrapped leaves are removed 2-5 days after hybridization. Lateral flower buds near the pod-filling area are removed 5-9 days later. Lateral flower buds near the pod-filling area are removed a second time 15-20 days later.

[0025] When the hybridization location is Hainan, the specific hybridization method is as follows:

[0026] A: Hybrid parent sowing: Sowing donor parents and recipient parents from different ecological zones in Hainan Province;

[0027] B: Light compensation measures: Provide supplemental lighting for parent plants from different ecological zones;

[0028] C: Temperature compensation measures: From the end of November to the beginning of February of the following year, when the temperature is below 20℃, cover the greenhouse to keep it warm and ensure that the temperature is ≥20℃ throughout the entire growth stage of the plant.

[0029] D: Integrated water and fertilizer management: Step A: Apply slow-release compound fertilizer as base fertilizer to the soil at the same time as soybean sowing. From the V3 stage of soybean, apply urea, amino acids, trace elements and growth regulators as top dressing; routine water management.

[0030] E: Hybridization technique: Parental hybridization is carried out between 9:00 and 15:00. The specific hybridization method is as follows: pollination is carried out at the same time as emasculation. At the moment of completion of pollination, the leaf wrapping operation is carried out. Hybridization is completed. The leaf wrapping is removed 2-5 days after the completion of hybridization. The lateral flower buds near the pod swelling flower are removed 5-9 days later. The lateral flower buds near the pod swelling flower are removed a second time 15-20 days later.

[0031] Preferably, when the hybridization location is Beijing, the time for emasculation in step E is 5:00-9:30 am, and the time for pollination is 9:30-11:30 am.

[0032] When the hybridization location is Hainan, the supplementary lighting in step B uses a light source with a specific light quality and the following spectral structure: 25% far-red light, 42% red light, 18% green light and 15% blue light.

[0033] The wavelength range of the far-red light is 730-750nm; the wavelength range of the red light is 640-680nm; the wavelength range of the green light is 530-560nm; and the wavelength range of the blue light is 460-490nm.

[0034] When the hybridization location is Hainan, in step B, the optimal light duration is 14-16 hours of light / 10-8 hours of darkness when the parent's ecological zone is in the southern region; 16-18 hours of light / 8-6 hours of darkness when the parent's ecological zone is in the Huang-Huai region; 18-22 hours of light / 6-2 hours of darkness when the parent's ecological zone is in the northeastern region; and 16-20 hours of light / 8-4 hours of darkness when the parent's ecological zone is in the northwestern region.

[0035] Preferably, the KASP molecular marker described in step (3) can be used for gene detection after soybean emergence and when the true leaves unfold. The KASP molecular marker labeling reaction system is as follows: 10 μL reaction system, 5 μL of 2× Master Mix, 0.01 μL of 100 μM forward primer 1, 0.01 μL of 100 μM forward primer 2, 0.03 μL of 100 μM reverse primer, 25-250 ng of DNA template, and ddH2O to make up to 10 μL.

[0036] Preferably, the KASP molecular marker reaction procedure in step (3) is as follows: 60℃ for 30 s; 95℃ for 10 min; 95℃ for 20 s, 61℃-55℃, -0.6℃ per cycle, annealing extension for 40 s, 10 cycles; 95℃ for 20 s, 55℃ annealing extension for 40 s, 30 cycles; then read the 60℃ for 30 s reading and perform KASP typing detection.

[0037] Preferably, the agronomic traits described in step (3) are specific phenotypic traits of the recipient parent.

[0038] Preferably, the reaction system of the SSR molecular marker in step (6) is as follows: 5 μL reaction system, 2.5 μL 2× PCR Mix, 0.1 μL 10 μM forward primer, 0.1 μL 10 μM reverse primer, 15-150 ng DNA template, and ddH2O to make up to 5 μL;

[0039] The reaction procedure for the SSR molecular marker is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 25 s, 32 cycles; extension at 72℃ for 10 min.

[0040] The number of SSR marker differences in backcrossing and breeding as specified in the "National Standards for the Approval of Genetically Modified Soybean Varieties (Trial)" in step (6) needs to be <2.

[0041] Another object of the present invention is to provide the application of the above-mentioned rapid directional backcrossing and breeding method for soybeans in the breeding of new soybean germplasm.

[0042] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The present invention uses 315 soybean varieties covering different ecological zones as recipient parents and glyphosate-resistant transgenic soybean Zhonghuang 6106 as donor parents for soybean directional backcrossing and conversion. In order to shorten the breeding time and screen superior varieties to the greatest extent, the hybridization method was improved in four aspects: light, temperature, water and fertilizer, and hybridization technology, which maximized the hybridization success rate and provided a large number of usable progeny for the subsequent backcrossing process. At the same time, screening was carried out in the hybrid progeny and backcross progeny. The screening method was improved by combining phenotypic screening and specific molecular marker screening, which significantly improved the screening efficiency, reduced the workload, shortened the breeding time to the greatest extent, and improved the breeding efficiency. The entire backcrossing and conversion process can be completed within 2 years, which is a significant improvement compared with the traditional method of 6 years. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0044] Figure 1 For: Shading control photoperiod facility for Beijing hybrid system.

[0045] Figure 2 The purpose of this study was to compare the significant differences in flowering time among different soybean varieties under different sowing conditions and light durations. A: Sowing in early April; B: Sowing in early June; Ns: No significant difference * < 0.01 ** < 0.001; *** < 0.0001; **** < 0.0000.

[0046] Figure 3 For: Comparison of the significant differences in flowering time of soybeans under different sowing dates with the same photoperiod; A: 18h light / 6h dark; B: 16h light / 8h dark; C: 12h light / 12h dark; D: 8h light / 16h dark; Ns: No significant difference *<0.01 **<0.001.

[0047] Figure 4 For: Beijing early spring seedling temperature control and insulation film covering technology, A: film covering device, B: seedling emergence effect.

[0048] Figure 5The following parameters are used to determine the impact of three different sowing times and temperatures on seedling emergence: A: Surface temperature at different sowing times; B: Number of days to emergence under different sowing conditions; C: Emergence rate under different sowing conditions. Note: * < 0.01 ** < 0.001, where dotted bars represent normal environment without mulching, and diagonal bars represent mulched sowing.

[0049] Figure 6 The purpose of this study was to compare the survival rates of hybrid fruits from 10 varieties under different management conditions. Note: The success rate comparison under both management models showed extremely significant differences.

[0050] Figure 7 The purpose of this study was to compare four optimized techniques for improving the survival rate of hybrid soybean fruits in four varieties in Beijing. Note: The comparison of the survival rate of hybrid fruits under the four treatments showed extremely significant differences.

[0051] Figure 8 For: Compensation and photoperiod regulation facility for Hainan soybean hybrid system.

[0052] Figure 9 The spectral structure ratio of light quality that promotes high-multiplication of soybeans.

[0053] Figure 10 For: Comparison of significant differences in flowering period and flowering duration of soybean under different light quality conditions, where A: flowering period, B: flowering duration; Ns: no significant difference *<0.01, **<0.001; ***<0.0001; ****<0.00001.

[0054] Figure 11 For: Comparison of significant differences in flowering time of soybeans under different photoperiods under customized light quality ratios. Note: Significance analysis of differences between different photoperiods and natural light 14h showed extremely significant differences.

[0055] Figure 12 The data represents the trends in the highest and lowest temperatures during the hybridization period of soybeans in Hainan.

[0056] Figure 13 The following are the effects of three different sowing times and temperatures on seedling emergence: A: Surface temperature at different sowing times; B: Number of days to emergence under different sowing conditions; C: Emergence rate under different sowing conditions. Note: * < 0.01, ** < 0.001.

[0057] Figure 14 The value is: survival rate of hybrid fruits of different varieties under temperature regulation; Note: The significant differences between the first stage and the second to fifth stages were all extremely significant.

[0058] Figure 15The purpose was to compare the survival rates of soybean hybrid fruits under five different hybridization treatments. Note: QX means emasculation the day before and pollination the day after; DTB means emasculation on the same day, uniform pollination without leaf covering; DTY means emasculation on the same day, uniform pollination with leaf covering; DSB means emasculation and pollination without leaf covering; DSY means emasculation and pollination with leaf covering. No significant differences were found in the survival rates of hybrid fruits under the five treatments. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] This study selected 315 bred varieties and superior lines covering different ecological zones of soybean cultivation in my country as recipient parents, and Zhonghuang 6106, a transgenic soybean tolerant to glyphosate herbicide, as the donor parent. The hybridization and backcrossing of these parents and their offspring were conducted at two locations: the Shunyi Experimental Station of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, Housangyuan Village, Zhaoquanying Town, Shunyi District, Beijing (116°57′27″E, 40°23′06″N), and the National Biological Breeding Zone of Nanbin Farm, Yazhou District, Sanya City, Hainan Province (109°19′19″E, 18°37′07″N).

[0061] Example 1

[0062] Research on hybridization methods in the Beijing area

[0063] (I) Photoperiod regulation technology affecting soybean hybrid survival rate in Beijing

[0064] A backcross breeding system was constructed using 315 soybean varieties (lines) from four ecological regions: southern China, Huang-Huai region, northeastern China, and northwestern China as recipient parents (Table 1) and the transgenic soybean Zhonghuang 6106 (provided by the Institute of Crop Science, Chinese Academy of Agricultural Sciences) as donor parent.

[0065] Table 1. Sources of 214 soybean varieties / lines

[0066]

[0067] Note: 212 representative varieties were selected from 315 soybean varieties, and their origins are listed.

[0068] Hybridization and backcrossing were conducted during this process. Based on Beijing's natural environmental factors, the goal was to ensure that varieties from all ecological zones could flower and mature normally in Beijing. This involved overcoming challenges such as completing at least two hybridizations during the planting period from April to November and achieving flowering coincidence between varieties from different ecological zones. Only in this way could the rapid backcrossing and breeding of soybeans be achieved. However, currently, some southern soybean varieties cannot mature due to excessively long daylight hours and long vegetative growth periods, preventing them from being induced into the reproductive growth stage. Furthermore, significant differences in flowering time between materials from different ecological zones greatly increase the difficulty of achieving flowering coincidence. To solve these problems, effectively improve the efficiency of soybean backcrossing and breeding, and increase the survival rate of hybrid soybean pods, we explored photoperiod regulation for varieties from different ecological zones. The Beijing hybridization system's shading control photoperiod facilities, such as... Figure 1 As shown, the specific research is as follows:

[0069] (1) Effects of photoperiod regulation on flowering period

[0070] Two representative sowing periods, early April and early June, were chosen to comprehensively and systematically investigate the length of the flowering period (emergence to initial flowering) of soybean varieties under four different photoperiods: 18 h, 16 h, 12 h, and 8 h. A detailed comparative study was conducted, and the experimental results are as follows: Figure 2 As shown.

[0071] Results Analysis: When sowing was carried out in early April, the results clearly showed that soybeans treated with 18-hour and 16-hour photoperiods had significantly longer flowering periods than those treated with 8-hour photoperiods. Further analysis of different varieties revealed that southern and Huang-Huai varieties also had significantly longer flowering periods under 18-hour and 16-hour photoperiods compared to those under 8-hour photoperiods.

[0072] Under the specific condition of 12-hour light, the characteristics exhibited by the Northeast varieties were not significantly different from those under the 8-hour light treatment. However, the Southern and Yellow River and Huai River varieties showed a unique pattern under the 12-hour photoperiod, with their flowering period being significantly longer than that under the 8-hour light condition.

[0073] (2) Effects of photoperiod regulation on flowering duration

[0074] Subsequently, the research focused on the flowering duration of soybean varieties, specifically the crucial stage from initial flowering to pod formation. The study found that the flowering duration of Northeast varieties remained essentially consistent under both 18-hour and 16-hour photoperiods, with no significant difference confirmed by rigorous statistical testing. This important finding implies that when cultivating Northeast varieties in the Beijing area, a mere 16-hour photoperiod is sufficient to meet the necessary conditions for flower organ development during hybridization. Furthermore, the relatively short flowering period is highly advantageous. This characteristic undoubtedly provides extremely favorable conditions for attempting two soybean hybridization experiments in the Beijing area.

[0075] For the Huang-Huai variety, a significant shortening of the flowering period was observed after 12 hours of light exposure; however, it is noteworthy that the reduction in flowering duration was not significant. A comprehensive and in-depth analysis of these two key factors—flowering period and flowering duration—also revealed that southern varieties could successfully achieve normal maturity under 8 hours of light. Furthermore, detailed analysis of the flowering period clarified that the flowering periods of southern varieties under 8 hours of light and Huang-Huai varieties under 12 hours of light are extremely similar. This similarity allows for successful overlap in their flowering periods, which is of great significance for soybean cultivation and hybridization research.

[0076] In summary, the study of flowering period in step (1) and the study of flowering duration in step (2) are as follows:

[0077] For soybean varieties from Northeast China, when the photoperiod is 16 hours, the flowering period is between 18 and 22 days. This flowering period is ideal, ensuring sufficient time for soybean hybridization. This allows for the completion of a single hybridization season within three months, which is of significant value in improving production efficiency in agricultural practice.

[0078] For the Huang-Huai variety, a photoperiod of 12 hours demonstrates an advantage in effectively shortening the growth period. Under these light conditions, the flowering period of soybeans is between 22 and 30 days. This flowering period also meets the requirement of completing a single-season hybridization within 3 months, providing favorable conditions for soybean hybridization.

[0079] For southern varieties, when the photoperiod is 8 hours, their flowering period is the same as that of Huang-Huai varieties under a 12-hour photoperiod treatment.

[0080] Based on the results of all the above experiments and various situations in actual production, in order to ensure that soybean varieties can be hybridized within the optimal hybridization time, maximize hybridization efficiency, accelerate the soybean hybridization process, and ultimately achieve the goal of completing two generations of hybridization in Beijing, the optimal photoperiods for the two sowing periods were finally determined: 16 h for Northeast varieties, 12 h for Huanghuai / Northwest varieties, and 8 h for Southern varieties.

[0081] It should also be noted that when sowing is in early April, the flowering period of Northeast, Northwest, Huang-Huai, and Southern varieties is approximately 38-42 days; when sowing is in early June, the flowering period is approximately 28-32 days. Therefore, the photoperiod limitation mentioned above in this invention maximizes the uniformity of flowering periods in different ecological regions, making them more consistent and maximizing the overlap of flowering periods. Furthermore, under the above photoperiod limitation, the flowering duration in each ecological region is relatively long, providing ample time for the application of hybridization technology. These results provide a highly valuable reference for the hybridization of soybean varieties in different ecological regions, powerfully promoting further development and exploration in related fields.

[0082] (3) In order to further explore the relationship between soybean growth and development and light, this invention conducted a comprehensive and detailed statistical analysis on the significance of flowering duration under the same light cycle but different sowing dates, that is, the time span from the initial flowering stage to the pod-setting stage. The results are as follows: Figure 3 As shown.

[0083] Results analysis, by Figure 3 It is evident that under long-day conditions, soybean flowering duration exhibits highly significant differences. These differences are not only observed among soybean varieties in different ecological zones but also particularly pronounced among different sowing dates for the same variety. As day conditions gradually shift towards short-day conditions, a clear trend emerges: the range of differences in soybean flowering duration between different sowing dates begins to narrow. Simultaneously, with the continuous reduction in day length, soybean flowering duration also shows a corresponding decrease. For example, under 8-hour day conditions, the flowering duration of soybean materials in Northeast China remains within a short range of 10 to 13 days; however, when the day length increases to 12 hours, the flowering duration extends to approximately 20 days. This demonstrates that the length of day length plays a crucial role in controlling the timing of soybean hybridization. Insufficient day length significantly shortens the available time for soybean hybridization, directly resulting in low hybridization efficiency and negatively impacting the survival rate of hybrid soybean pods.

[0084] (II) Determination of Temperature Compensation Measures

[0085] To increase the number of backcrosses of soybeans to meet experimental needs, two hybridization seasons need to be completed in the Beijing area between April and November. Based on this requirement, soybean planting must begin in early April each year. However, temperatures in Beijing in early April typically fluctuate between 4-18℃, which affects soybean emergence, significantly prolonging the emergence time and posing a risk that some soybean seeds may fail to germinate. This severely impacts the efficiency of soybean hybridization.

[0086] To accomplish the soybean hybridization task more efficiently, two different treatment methods were designed:

[0087] (1) Post-planting mulching and heat preservation treatment

[0088] (2) Allow soybeans to grow normally after planting.

[0089] A comparative study was conducted on soybeans sown at different dates from April to June under both mulching and natural field conditions. Soil surface temperature, emergence days, and emergence rate were measured. The experimental results are as follows: Figure 4 and Figure 5 As shown.

[0090] Results Analysis: The results show that mulching has a significant effect on increasing soil surface temperature. On average, mulching can raise the soil surface temperature by approximately 4°C. Regarding emergence time, when soybeans are sown in early April, mulching allows for earlier emergence by 6 days, saving more than 6 days compared to natural field conditions. When sown in early May, mulching can save more than 3 days for soybean emergence. And when sown in June, mulching can advance soybean emergence by 1 day.

[0091] From the perspective of germination rate, mulching significantly improves the germination rate by approximately 40% when sowing in early April. When sowing in May, mulching further increases the germination rate by over 30%. However, the situation is different when sowing in June; mulching causes a decrease in germination rate of over 50%. Field observations revealed that mulching in June causes excessively high temperatures in a short period, which deactivates the seeds and severely impacts the germination rate.

[0092] Based on the above findings, it can be determined that using mulching in April and May is an effective method to help soybean seedlings emerge. However, when outdoor temperatures rise above 20°C, mulching carries serious risks and may adversely affect soybean seedling emergence. These research results provide important practical reference for soybean planting at different stages in the Beijing area, helping to rationally plan planting measures and improve the efficiency of soybean hybridization.

[0093] (III) Determination of integrated water and fertilizer management measures

[0094] To investigate the effects of different water and fertilizer management practices on hybridization survival rates, two sets of comparative experiments were carefully designed:

[0095] (1) Integrated water and fertilizer management: Hybrid parents are sown in pots, with a pot height of 35cm and a diameter of 45cm, and 6-8 soybean plants are planted in each pot. The potting soil is a mixture of nutrient substrate, vermiculite and field soil in a ratio of 3:1:3. 0.1 kg of slow-release compound fertilizer is added as base fertilizer per 100 kg of mixed soil. From the soybean V3 stage, urea is added every 10 days according to the plant growth, with a concentration of 0.01 kg / pot for foliar spraying. Amino acids and trace elements are sprayed every 7 days, and brassinolide is sprayed every 15 days. The amount of each fertilizer is added according to the conventional amount. Conventional water management is also performed.

[0096] (2) Traditional field planting management: watering during the sowing period to promote seedling emergence, pest control in the field 3-5 times, applying 80 catties of compound fertilizer per mu before planting, cultivating once during the 4-5 leaf stage, and weeding 2-3 times throughout the entire growth period.

[0097] A comprehensive and detailed comparative study was conducted in a real field environment. Several representative soybean varieties were selected as research subjects. Through rigorous statistical analysis of the key indicator of soybean hybrid pod formation rate, the study clearly and intuitively demonstrated the specific impact of two drastically different management models on the survival rate of soybean hybrid pods. The experimental results are as follows: Figure 6 As shown.

[0098] Results Analysis: Under the positive effects of integrated water and fertilizer management, soybean plants exhibited a significant overall growth trend. Regarding the important growth indicator of pod number, the number of pods produced by soybean plants under integrated water and fertilizer management was significantly higher compared to traditional normal field management conditions. In the specific experiment, simultaneous comparative experiments were conducted on 10 carefully selected soybean varieties under both field management and integrated water and fertilizer management conditions. Based on a standardized experimental setup that strictly ensured the same hybridization time and the same number of hybrid flowers, the pod formation of each variety was accurately statistically analyzed, and the survival rate of the hybrid pods was calculated using scientific methods. Rigorous data comparison and analysis revealed that the survival rate of the hybrid pods of these 10 varieties under integrated water and fertilizer management conditions was significantly higher than the corresponding values ​​under field management conditions.

[0099] (iv) Determination of hybridization techniques

[0100] In the complex and delicate process of soybean hybridization, weather conditions are undoubtedly a crucial and highly variable factor. During hybridization, changes in weather conditions, such as sunshine, cloudy skies, and fluctuations in temperature and humidity, can directly or indirectly affect the state of soybean flower organs and the effectiveness of hybridization operations. Furthermore, the complex structure and small size of soybean flower organs make automated mechanization difficult, necessitating manual demasking and pollination. In this process, the number of operators and their skill level largely determine the success rate of hybridization. Therefore, while actively exploring ways to modify the external environment to facilitate hybridization, focusing on improving the skill level of manual operations and the fruit set rate of hybrid fruits has become a core focus and key direction for improving soybean hybridization technology.

[0101] For the reasons mentioned above, this invention has conducted in-depth exploration and experimentation on soybean hybridization methods, and set up the following experimental groups:

[0102] (1) Go to the male one day in advance for pollination the next day (go to the male in advance);

[0103] (2) Males are removed on the same day (5:00 - 9:30), and pollination is carried out on the same day (9:30 - 11:30) without covering the leaves (males are removed and pollination is carried out without covering the leaves);

[0104] (3) Emasculation on the same day (5:00 - 9:30), pollination on the same day (9:30 - 11:30) and leaf wrapping (emasculation, pollination and leaf wrapping);

[0105] (4) Emasculation and pollination should be carried out simultaneously with leaf wrapping (emasculation and pollination should be carried out immediately with leaf wrapping).

[0106] To ensure the scientific validity and representativeness of the research results, four soybean varieties with different genetic characteristics, growth habits, and regional adaptability were carefully selected. They underwent meticulous treatment and comprehensive comparative studies. The experimental results are as follows: Figure 7 As shown.

[0107] Results analysis: such as Figure 7 The operation of immediately wrapping the leaves after emasculation and pollination, as shown, can significantly improve the survival rate of hybrid fruits and is the optimal hybridization operation. The specific reasons are analyzed as follows:

[0108] The emasculation of soybean flowers is performed between 5:00 AM and 9:30 AM. During this period, the physiological state of the soybean flower organs is relatively stable, and cell activity is at an appropriate level for emasculation, which can minimize the damage and stress caused to the flowers by the emasculation process.

[0109] Between 9:30 and 11:30, while the flower stigmas are fresh, highly active, and the external environmental conditions are relatively stable, pollination should be carried out quickly.

[0110] Immediately after pollination, the leaves should be wrapped around the stigma. This timely action has multiple important meanings and significant benefits. From a light-shading perspective, it effectively blocks direct sunlight, creating a relatively soft and stable light environment for the hybridization site. Especially during periods of intense sunlight, the ultraviolet radiation is high. Without protection, it can easily cause irreversible damage to the stigma and the entire pollination structure, such as sunburn and aging of the stigma cells, thus affecting pollen germination and fertilization. The wrapping leaves act like a natural "sunshade," protecting the hybridization process and ensuring it proceeds smoothly under suitable light intensity. Furthermore, in practice, to maintain the water balance and physiological activity of the hybrid flowers, timely watering is often necessary. However, without the protection of the leaves, water droplets can easily drip onto the stigma due to gravity. These seemingly tiny water droplets, due to their surface tension and potential impurities, can cause mechanical damage or infection to the stigma, thus interfering with the normal pollination and fertilization process. The calyx acts as a reliable "shield," cleverly shielding these potential risks and effectively ensuring the integrity and smoothness of the hybridization and pollination process.

[0111] It is worth noting that hybridization is not limited to the treatment at the moment of pollination. The subsequent management measures after hybridization also play a crucial role in the whole process and should not be ignored.

[0112] Two days after hybridization, precisely removing the wrapping leaves becomes a crucial task. At this point, the hybridized parts, after two days of protection and adaptation, have initially developed a certain ability to resist changes in the external environment. Removing the wrapping leaves allows the hybridized flowers to gradually come into contact with natural light, air circulation, and other environmental factors, promoting the normal development of physiological processes such as photosynthesis and respiration, and helping them smoothly transition from the artificially protected state to the natural growth and development stage.

[0113] Five days after hybridization, lateral flower buds near the pod's enlarging shape begin to grow and develop. If left unchecked, these lateral buds will engage in fierce competition for nutrients with the hybrid pod. Since the hybrid pod carries the important mission of cultivating superior new varieties, its nutritional needs are more urgent and demanding. Therefore, these lateral flower buds must be carefully removed to ensure that the hybrid pod can preferentially access sufficient nutrient resources, guaranteeing its normal enlargement, growth, and development.

[0114] Fifteen days after hybridization, a second round of lateral flower bud removal is performed, and any self-pollinating pods that may appear on the nodes above and below the pod-bearing section are completely removed. This is because as the hybrid pods grow, the lateral flower buds around them may regrow, and the self-pollinating pods may also consume some nutrients. Through this series of meticulous and systematic management measures, it is possible to ensure that the hybrid pods are in an ideal state of sufficient and stable nutrient supply throughout their growth and development, effectively preventing flower and pod drop, and building a solid and reliable guarantee for the successful development of soybean hybrid fruits and ultimately achieving a high survival rate.

[0115] In summary, this comprehensive hybridization technology operation process and management strategy is a valuable achievement derived from extensive practical verification and scientific research. It not only provides a practical technical path and operational standards for optimizing soybean hybridization technology, but also offers invaluable practical reference for its wider application. It is expected to trigger a wave of technological innovation and progress in the field of soybean breeding, playing a crucial leading and supporting role in cultivating more high-yielding, high-quality, and stress-resistant new soybean varieties, and promoting the sustainable development and upgrading of the soybean industry.

[0116] Based on the above experiments, the hybridization method for soybean varieties from different ecological zones in Beijing was successfully determined, as follows:

[0117] A method for improving the survival rate of soybean hybrids from different ecological zones in Beijing includes the following steps:

[0118] (1) Sowing of hybrid parent lines: 315 parent materials from four ecological regions, namely the southern region, the Huang-Huai region, the northeast region and the northwest region, were used as recipient parents, and the transgenic soybean Zhonghuang 6106 was used as the donor parent. They were sown in pots with a height of 35cm and a diameter of 45cm. The potting soil was a mixture of nutrient substrate, vermiculite and field soil in a ratio of 3:1:3. The soil filling height of the pots was uniform, 7cm from the edge of the pot. After filling the soil, 2cm of vermiculite was laid on top. The soybeans were planted on the vermiculite and covered with 3-4cm of soil. Water was sprayed lightly.

[0119] (2) Photoperiod adjustment: The photoperiod of soybeans in different ecological zones was adjusted to 8h for soybean varieties in the south; 12h for soybean varieties in the Huang-Huai region; 16h for soybean varieties in the Northeast region; and 16h for soybean varieties in the Northwest region.

[0120] (3) Temperature compensation measures: In Beijing, after soybeans are sown in April and May each year, they are covered with film. When the outdoor temperature is ≥20℃, the film covering is stopped. In order to avoid the impact of low temperature environment on the growth of soybeans during the flowering period, a shading arch is built by combining opaque reflective film with galvanized pipes, and artificial shading is used to control the photoperiod.

[0121] (4) Integrated water and fertilizer management: Step (1) When sowing soybeans, apply slow-release compound fertilizer as base fertilizer to the mixed soil. The amount of fertilizer is 0.1 kg per 100 kg of mixed soil. At the same time, water the bottom thoroughly. From the V3 stage of soybeans, add urea every 10 days according to the plant growth. The concentration of the addition is 0.01 kg / pot for foliar spraying. Spray amino acids and trace elements every 7 days and brassinolide every 15 days. The amount of each fertilizer added can be added according to the conventional amount. Conventional water management.

[0122] (5) Hybridization technique: remove males from 5:00 to 9:30 on the same day, pollinate from 9:30 to 11:30 on the same day, wrap the leaves immediately after pollination, and the hybridization is completed. Remove the wrapping leaves 2 days after the hybridization is completed, and remove the lateral flower buds near the pod swelling flower 5 days later; remove the lateral flower buds near the pod swelling flower 15 days later.

[0123] Example 2

[0124] Research on hybridization methods in Hainan

[0125] (I) Regulation techniques for light compensation measures affecting the survival rate of hybrid soybeans in Hainan

[0126] Construction of a supplemental lighting greenhouse hybridization platform

[0127] To prolong flowering time, enlarge flower organs to reduce hybridization difficulty and improve hybridization efficiency, a light quality ratio design was implemented based on the light requirements and growth conditions of different soybean tissues and organs. Supplemental lighting greenhouses were constructed using galvanized iron pipes, each approximately 49.0 square meters (7.0m × 7.0m). To increase illumination duration, waterproof LED tubes (manufactured by Ningbo Yanghui Instrument Co., Ltd.) were used as nighttime supplemental lighting, suspended by high-load-bearing steel wire. Six tubes were suspended in each greenhouse, with six 1.2m plots planted, spaced 0.5m apart. The light source was positioned 2.5 meters above the ground, equipped with a free-lifting device and a timer control. The Hainan soybean hybrid system's photoperiod compensation and regulation facilities are as follows. Figure 8 As shown.

[0128] (1) Comparison experiment of different light sources

[0129] Using Hainan (with relatively short average sunshine hours, around 14 hours per day) as the experimental base, supplemental lighting experiments were conducted. Three control experiments were set up during the experiment: a customized light source with specific light quality ratios, an incandescent lamp source, and natural field lighting, as follows:

[0130] Experimental Group 1: A customized light source with a spectral structure of 25% far-red (730-750nm), 42% red (640-680nm), 18% green (530-560nm), and 15% blue (460-490nm). The supplemental lighting duration was 18 hours, the lamp power was 38W, and the illumination control range was 200-30 μmol / m².s. The spectral structure ratio was as follows: Figure 9 As shown;

[0131] Experimental Group 2: Incandescent lamp light source, supplemental lighting duration 18h, lamp power 100W;

[0132] Experimental group 3: Natural field light, 14h.

[0133] This study compared the flowering time and floral organ status of soybeans in Hainan under three different light conditions. Specific observation indicators included recording the flowering period and duration of soybeans, as well as visually assessing the flowering status. The experimental results are as follows: Figure 10 As shown in Table 2.

[0134] Table 2. Soybean flower organ status under different light quality conditions

[0135]

[0136] Results Analysis: Figure 10 As shown in Table 2, an 18-hour light exposure setting has a certain promoting effect on the hybridization of Northeastern varieties in Hainan. However, some negative phenomena occurred for Huang-Huai and Southern varieties. Specifically, the supplemental lighting time was too long, leading to a prolonged flowering period. Under these circumstances, soybeans exhibit a reverse growth state, that is, they revert from the early stage of reproductive growth to a new vegetative growth stage. This reverse growth results in an increase in the number of flowers, but smaller flower organs. More seriously, the soybean flower organs cannot open normally, which greatly affects the survival rate of soybean hybrid fruits.

[0137] Meanwhile, the study also found that using incandescent lamps for supplemental lighting did not have a significant impact on the size of flower organs, the degree of opening, or the duration of flowering, nor did it improve the survival rate of hybrid fruits. Therefore, experimental group 1 was selected to customize the light quality ratio light source for further in-depth research.

[0138] (2) Comparison experiment of different light compensation times

[0139] To further explore the suitable light compensation time for soybean varieties in different ecological zones under customized light quality ratios, this study set up different supplemental lighting times, specifically 16h, 18h, 22h, and 24h. By analyzing the opening status of floral organs (which directly relates to the difficulty of soybean hybridization) and flowering duration (which determines the time available for soybean hybridization), the experimental results are as follows: Figure 11 As shown in Table 3.

[0140] Table 3. Comparison of soybean flower organ opening status under customized light quality ratio light source conditions with different photoperiods.

[0141]

[0142] Results Analysis: The suitable light duration varies among varieties in different ecological zones. Excessive light duration is counterproductive for Huang-Huai / Southern varieties. Therefore, supplemental lighting time needs to be adjusted according to the variety's origin in different ecological zones. Considering various factors, this invention concludes from Table 1 that for Northeastern varieties (Northern spring soybeans), 16-24 hours of supplemental lighting is appropriate. Figure 4 (The longer the flowering period, the better) The conclusion drawn is 16-24 hours. Considering that too long a light exposure time will lengthen the growth period, and to save time, 22 hours is sufficient to meet the hybridization requirements, so the time was shortened from 24 hours to 22 hours. Therefore, 18-22 hours is determined to be the optimal light exposure time for hybridization for Northeast varieties; for Northwest varieties, considering various factors, 16-18 hours is more suitable; the optimal light exposure time for Huanghuai varieties is 16-18 hours; and for Southern varieties, the conclusion drawn from Table 1 is 16-24 hours. Figure 4 The conclusion drawn from the study on flowering duration (longer is better) was 16-24 hours. Since 14 hours is the normal light duration in Hainan, which southern materials can complete, considering the overall experimental time, 14-16 hours was determined to be the optimal light duration for hybridization of southern varieties. These research findings will provide important reference for light duration in soybean hybridization work in Hainan, helping to improve the success rate of soybean hybridization.

[0143] (II) Determination of Temperature Compensation Measures

[0144] During the practice of soybean hybridization in Hainan, detailed monitoring and analysis of temperature data revealed that, periodically from late November to early February of the following year, there are two periods of consistently low temperatures, with short-term temperatures dropping below 20°C. Figure 12 As shown.

[0145] This invention addresses this specific temperature phenomenon by conducting in-depth statistical analysis of soybean emergence rate and hybrid seedling survival rate at different stages. The experimental results are as follows: Figure 13 and Figure 14 As shown.

[0146] Results Analysis: The research results clearly demonstrate a strong positive correlation between temperature and the survival rate and emergence rate of soybean hybrid fruits. With the gradual increase in temperature, the survival rate of soybean hybrid fruits showed a significant improvement. The increase was particularly significant for hybrid fruits from southern regions, jumping from a relatively low 2% to 60%; the survival rate of hybrid fruits from northeastern regions also increased significantly from 30% to 60%. This definitively demonstrates that temperature plays a crucial and decisive role in improving the survival rate and emergence rate of soybean hybrid fruits. Therefore, this invention ultimately determines that when the temperature in Hainan is below 20℃, a greenhouse structure should be used to raise the temperature to ensure it remains above 20℃.

[0147] (III) Determination of integrated water and fertilizer management measures

[0148] Same as Example 1.

[0149] (iv) Determination of hybridization techniques

[0150] In Hainan's unique climate, soybean hybridization faces numerous challenges. Hainan experiences intense ultraviolet radiation and often unbearably hot weather during pollination, all of which severely threaten the survival rate of hybrid soybean pods. To effectively improve the survival rate of hybrid soybean pods, this invention carefully designed five different treatment methods and selected 12 soybean varieties with different characteristics for hybridization experiments. The survival rate of the hybrid pods was then precisely statistically analyzed, and the experimental results are as follows: Figure 15 As shown.

[0151] The five hybridization methods are as follows:

[0152] 1: Remove males the day before and pollinate the next day, without covering the leaves (QX);

[0153] 2: Males are removed on the same day, and pollination is carried out uniformly without leaf covering after male removal (DTB).

[0154] 3: Remove males on the same day, and then pollinate and wrap leaves uniformly (DTY).

[0155] 4: Pollination without leaf wrapping during emasculation (DSB);

[0156] 5: Pollinate and cover the leaves simultaneously during emasculation (DSY).

[0157] Results Analysis: Figure 15The study indicates that leaf-wrapping treatment has a significant positive effect on improving the survival rate of hybrid soybean pods. Among these treatments, the method of immediately wrapping the pods after emasculation and pollination yields the most ideal results. Compared to the method of not wrapping the pods and pollinating the following day, the survival rate of the hybrid pods can be increased by more than 15%. In-depth analysis and speculation suggest that this is most likely due to the extremely strong ultraviolet radiation in Hainan, where soybean hybridization is carried out, causing severe damage to the soybean stigmas. Detailed observation of the stigma condition on the second day after emasculation revealed that if no shading protection is provided within 15 minutes after emasculation, the stigmas will noticeably darken and become damaged. This undoubtedly greatly hinders the subsequent pollination and fertilization process, thus reducing the survival rate of the hybrid pods.

[0158] Furthermore, Hainan's unique climate is characterized by severe nighttime dampness, leading to high humidity. This high humidity makes soybean pollen sticky and heavy, severely hindering pollen dispersal and hybridization. Multiple field observations and data analysis revealed that after 9:00 AM, as the temperature gradually rises, the humidity dissipates, resulting in optimal pollen dispersal and providing favorable conditions for pollination. However, after 3:00 PM, pollen dispersal deteriorates over time, hindering successful hybridization.

[0159] Based on the above research results and analysis, in order to maximize the survival rate of hybrid berries when conducting soybean hybridization in Hainan, the hybridization operation is generally completed between 9:00 AM and 3:00 PM. The specific procedure involves emasculation and simultaneous pollination, and leaf shading must be performed immediately after pollination. This is to ensure that the stigma is not exposed to direct sunlight for an extended period, preventing sunburn and effectively guaranteeing the survival rate of the hybrid berries. This provides a scientific basis and technical guidance for the smooth implementation of soybean hybridization breeding in Hainan.

[0160] Based on the above experiments, the hybridization method for soybean varieties from different ecological zones in Hainan was successfully determined, as detailed below:

[0161] A method for improving the hybrid survival rate of soybeans from different ecological zones in Hainan includes the following steps:

[0162] (1) Sowing of hybrid parent lines: 315 parent materials from four ecological zones, namely the southern region, the Huang-Huai region, the northeastern region and the northwestern region, were used as recipient parents, and the transgenic soybean Zhonghuang 6106 was used as the donor parent. They were sown in pots with a height of 35cm and a diameter of 45cm. The potting soil was a mixture of nutrient substrate, vermiculite and field soil in a ratio of 3:1:3. The soil filling height of the pots was uniform, 7cm from the edge of the pot. After filling the soil, 2cm of vermiculite was laid on top, and the soybeans were planted on the vermiculite. The soil was covered with 3-4cm and watered lightly.

[0163] (2) Light compensation measures: A light source with specific light quality (spectral structure of far-red light (730-750nm) 25%, red light (640-680nm) 42%, green light (530-560nm) 18% and blue light (460-490nm) 15%, supplemental lighting duration of 18h, lamp power of 38W, light control range: 200-30μmol / m².s) was used to supplement the parent plants sown in step (1); for varieties in the southern ecological zone, the optimal light time is 14-16h; for varieties in the Huanghuai ecological zone, the optimal light time is 16-18h; for varieties in the northeastern ecological zone, the optimal light time is 18-22h; for varieties in the northwestern ecological zone, the optimal light time is 16-18h.

[0164] (3) Temperature compensation measures: In Hainan, from the end of November to the beginning of February of the following year, in order to avoid the impact of low temperature environment on the growth of soybeans during the flowering period, galvanized pipes are used to build arched greenhouse facilities, and horticultural standard height light-transmitting non-drip film is used to cover the greenhouse for heat preservation. The greenhouse is 1.5m high and 2m wide. The heat preservation time is from 17:00 in the evening to 8:00 the next day to ensure that the temperature of the plant growth environment is controlled above 20℃ during the low temperature period.

[0165] (4) Integrated water and fertilizer management: Step (1) When sowing soybeans, apply slow-release compound fertilizer as base fertilizer to the mixed soil. The amount of fertilizer is 0.1 kg per 100 kg of mixed soil. At the same time, water the bottom thoroughly. From the V3 stage of soybeans, add urea every 10 days according to the plant growth. The concentration of the addition is 0.01 kg / pot for foliar spraying. Spray amino acids and trace elements every 7 days and brassinolide every 15 days.

[0166] (5) Hybridization technique: The parent hybridization is carried out from 9:00 to 15:00. The specific hybridization method is as follows: pollination is carried out at the same time as emasculation. At the moment of completion of pollination, the leaf wrapping operation is carried out. After the hybridization is completed, the leaf wrapping is removed 2-5 days later. After 5-9 days, the lateral flower buds near the pod swelling flower buds are removed. After 15-20 days, the lateral flower buds near the pod swelling flower buds are removed again.

[0167] This invention's research on improving hybrid survival rates was first conducted in Hainan, then in Beijing, and involved multiple in-depth studies and experiments during breeding practices in Hainan. In the first year, active participation in hybridization work was undertaken, with five phases of hybridization trials meticulously planned and completed. Initially, due to the lack of research on supplemental lighting in the Hainan region, a uniform supplemental lighting method was adopted, failing to fully consider the differences in the ecological zones of different varieties. This resulted in excessively long supplemental lighting periods for Huanghuai and southern varieties. This unreasonable light duration triggered flower reversal, leading to a dismal hybrid fruit survival rate of 0%. This result undoubtedly presented a significant challenge to the research, but also provided valuable lessons for subsequent adjustments.

[0168] Entering the second phase of hybridization experiments, the research team astutely realized that photoperiod duration might be a key factor affecting the survival rate of hybrid fruits, and thus attempted to adjust the photoperiod. However, unfavorable weather conditions arose; the temperature was too low, severely impacting pollen activity. Despite optimization of photoperiod, the adverse environmental factor of low temperature still limited the success rate of hybridization, resulting in a survival rate of less than 2% for the hybrid fruits. Faced with this predicament, the research team did not lose heart but instead deeply analyzed the root causes of the problem and decided to address it from multiple perspectives.

[0169] In subsequent experiments, especially the fourth phase (December 2022 - May 2023), the research team fully absorbed the lessons learned from previous experiments and adopted more precise strategies. Based on the different ecological zones of the varieties, precise supplemental lighting measures were implemented, tailoring the most suitable light duration for each variety to ensure normal photosynthesis and growth. Simultaneously, temperature control was achieved using insulated greenhouses, effectively resisting the interference of low external temperatures and creating a relatively stable and suitable environment for soybean hybridization. These efforts finally yielded significant results, with the hybridization success rate greatly increasing to 50%. To further verify the reliability and stability of this achievement, a fifth verification experiment was conducted in Hainan in 2023 (corresponding to the technical solution defined above in this invention). The results were encouraging, with the survival rate of hybrid fruits significantly increasing to 65%, fully demonstrating that the technical system constructed in this invention has a practical and feasible effect in improving the survival rate of hybrid fruits.

[0170] In Beijing's hybridization nurseries, the survival rate of hybrid fruits has long been plagued by external environmental factors, resulting in significant fluctuations and instability. However, with the gradual establishment and improvement of the backcross breeding technology system, this situation has been effectively improved. Based on the successful experience in Hainan, and combined with the actual environmental characteristics of Beijing, the technology system was adaptively adjusted and optimized. Through these efforts, the survival rate of soybean hybrid fruits in Beijing has steadily increased from a relatively low average of 37% to over 60%. In particular, in 2024, by fully utilizing the constructed system technology, the survival rate of hybrid fruits successfully and stably exceeded the previous level, reaching 67%, demonstrating the strong adaptability and effectiveness of this technology system under different environments (Table 4).

[0171] Table 4. Success rates of hybrids bred using the backcrossing and conversion system.

[0172]

[0173] Example 3

[0174] Backcross breeding and targeted selection

[0175] Based on the hybridization in Examples 1-2, targeted screening was performed on the F1 generation and backcross progeny. The specific screening process is as follows:

[0176] (a) Phenotypic targeted screening

[0177] (1) Glyphosate tolerance identification: Glyphosate tolerance was identified in F1 generation and backcross progeny. The identification time was when the first compound leaf was fully expanded. The identification method was glyphosate directional spraying. The identification concentration was 200 ml / mu of glyphosate isopropylamine salt (Roundup). About 30% of false hybrids and non-resistant offspring could be eliminated by glyphosate spraying.

[0178] (2) Screening of agronomic traits: Referring to the "Description Specifications and Data Standards for Soybean Germplasm Resources" edited by Qiu Lijuan et al., the agronomic traits of the recurrent parents were investigated. Plants with flower color, leaf shape, pubescence color, growth period, or other special phenotypes consistent with the description of the recurrent parents were selected from the hybrid or backcross progeny for subsequent backcrossing. For example, taking Zhonghuang 30 as an example, when it was used as the recipient parent for hybridization and backcrossing with Zhonghuang 6106, the sparse pubescence of Zhonghuang 30 could be used as one of the agronomic trait screening indicators. Progeny with sparse pubescence were selected from the hybrid and backcross progeny for subsequent operations. Another example is Qihuang 34, which has large leaves with raised, vesicular structures. Table 5 lists the specific agronomic traits of 25 recurrent parents.

[0179] Table 5 Phenotypes of agronomic traits in recurrent parents

[0180]

[0181] (ii) Genotyping

[0182] (1) KASP molecular marker screening

[0183] The KASP molecular marker is application number: 202511152773.6, patent name: A KASP marker for detecting Huang 6106 in genetically modified soybeans and the ZH6106-2R and ZH6106-3R KASP molecular markers mentioned in its application.

[0184] DNA was extracted from the samples using a DNA extraction kit (Kangwei Century CW2361S). DNA quality was checked and template concentration adjusted using a NanoDropEight spectrophotometer. A 10 μL reaction mixture was prepared for each well (Table 6). Primers were KASP marker primers designed specifically for the GAT gene insertion site.

[0185] Table 6 KASP reaction system (10 μL reaction system)

[0186]

[0187] On the QuantStudio 7 Pro, the reaction program was as follows: pre-reading at 60℃ for 30 s; pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s, 61℃-55℃, -0.6℃ per cycle, annealing extension for 40 s, 10 cycles; denaturation at 95℃ for 20 s, annealing extension at 55℃ for 40 s, 30 cycles; post-reading at 60℃ for 30 s for KASP typing.

[0188] In research related to soybean hybridization breeding, timing is a crucial factor. Whether in regions with unique climatic conditions like Hainan or in environments like Beijing, to achieve the important goal of completing two hybridizations per year within a short period, it is essential to rationally regulate the soybean growth cycle. Shortening the flowering period, and consequently the maturity period, is one of the key strategies.

[0189] However, in practice, we face a thorny problem. Genotyping, a crucial step in accurately determining hybridization and selecting superior individual plants, typically takes more than 25 days. But in some special cases in Hainan and Beijing, some soybean materials have shorter flowering periods; the entire time from the unfolding of the first compound leaf to flowering is less than 25 days. This makes genotyping according to the conventional procedure extremely time-sensitive, making it difficult to complete the identification process during the critical flowering stage, potentially impacting the entire hybridization breeding process.

[0190] To effectively address this challenge and ensure successful genotyping at flowering time, this invention specifically develops the KASP marker technology for targeted identification of the target gene GAT. Using this molecular marker, sampling and identification can be conducted immediately after soybean emergence, when the true leaves have just unfolded, eliminating the need for reliance on glyphosate spraying for identification (which has limitations. Firstly, glyphosate spraying can only be performed after the first compound leaf of the soybean has fully unfolded, a process that itself takes 10-15 days. Secondly, even after glyphosate spraying, phenotypic manifestation requires an additional 3-5 days. Therefore, from the unfolding of the first compound leaf to the point where the phenotype after glyphosate spraying can be used to determine the situation, a total of approximately 13-20 days are needed. In some soybean materials from Hainan or Beijing, the time from the unfolding of the first compound leaf to flowering is even less than 15 days. This means that if glyphosate spraying phenotypic identification is performed first, followed by sampling and background recovery rate testing, the time available for subsequent molecular testing will be severely insufficient (less than 15 days). (This obviously cannot meet the time requirements for genotyping identification to determine true and false hybridization and resistance.) Compared to the previous process that relied solely on glyphosate spraying for identification, we can complete sampling and testing 15-17 days earlier. This measure can effectively shorten the entire identification process by at least 15 days. Through this time optimization, we can effectively ensure that key genotyping identification work such as background chip sequencing is completed before flowering, thus providing strong technical support for precision-assisted backcrossing and breeding. This ensures that the entire soybean hybridization breeding work can proceed smoothly according to the predetermined plan, improves breeding efficiency, and lays a solid foundation for cultivating higher-quality soybean varieties.

[0191] In addition, although there are some detection markers for the GAT gene that can be used for identification, these existing markers are mainly implemented by agarose testing. This detection method is relatively inefficient in practical applications and cannot meet our urgent need for efficient and rapid detection in the work of completing hybridization breeding in a timely manner.

[0192] (2) Identification of Zhongdouxin No. 1 chip (The Zhongdouxin No. 1 chip used in this invention is the chip in patent number 202180002016.4)

[0193] DNA was extracted from the samples using a DNA extraction kit (Kangwei Century CW2361S). The gDNA content of the samples was quantified using a Nanodrop ND-2000 (Themro Scientific) combined with agarose gel electrophoresis, and the samples were diluted to 50 ng / L. First, whole-genome amplification was performed on the identified samples. The amplification was carried out at 37℃ for 20–24 h. Then, the gDNA was fragmented, precipitated, and resuspended. The resuspended DNA fragments were added to a microarray for hybridization and incubated at 48℃ for 16–24 h. After hybridization, non-specifically bound DNA was washed away, and single-base extension was performed on the remaining specifically bound sites. After staining, the DNA was scanned using an Illumina iScan Reader.

[0194] The recurrent parent and the genotypes of glyphosate-tolerant single plants with similar comprehensive agronomic traits to the parent were identified using the Zhongdouxin No. 1 chip. 1.69 million SNP loci data were obtained for each sample. The similarity between the recurrent parent and the hybrid offspring was compared by using the IF function of EXCEL to obtain the similarity parameters between the offspring and the recurrent parent, which served as the background recovery rate index.

[0195] In traditional backcrossing breeding strategies, if single-gene iterative algorithms are used for calculation, a significant amount of resources are required in each generation to ensure that the backcross offspring are identical to or have only minimal differences (less than 2 SSRs) with the recurrent parents. Specifically, backcrossing operations need to be performed on 50 offspring plants in each generation, and the entire backcrossing process is lengthy, requiring more than 6 backcrossing generations (Table 7). This traditional method is not only extremely labor-intensive, consuming a large amount of manpower, resources, and time, but also has significant limitations in efficiency, making it difficult to meet the urgent needs of modern agricultural breeding for speed and precision.

[0196] To overcome this bottleneck, this invention focuses on reducing workload, decreasing reliance on the number of individual plants, and shortening backcross generations, thereby accelerating the backcrossing process and achieving precise backcrossing. Based on this, building upon the fundamental step of phenotypic identification, we fully utilize the powerful capabilities of the Zhongdouxin No. 1 chip, independently developed by the applicant, to conduct comprehensive and in-depth genotypic identification of backcross progeny and recurrent parents. By precisely analyzing the SNP similarity ratio between progeny and recurrent parents, a scientific and efficient screening system is constructed. In each backcross generation, based on this screening system, the 5-7 individual plants with the highest recovery rate are precisely selected from numerous progeny. Extensive experimental verification and data analysis have revealed that these 5-7 individual plants can fully represent the characteristics of more than 50 samples, exhibiting high representativeness and stability in genetic information and phenotypic features (Table 8).

[0197] Table 7 Iterative Algorithm for Background Recovery Rate of Single-Gene Backcross Transformation (Theoretical Backcross Generations and Number of Plants)

[0198]

[0199] Table 8. Background recovery rate detection percentage of 50 backcross progeny plants of 26 varieties

[0200]

[0201] This innovative molecular marker screening strategy has yielded remarkable results. From a workload reduction perspective, it effectively reduces hybridization work by over 70%. This means that with the same time and resource investment, researchers can process more backcross combinations, significantly increasing the throughput of breeding work. Considering labor cost savings, due to the substantial reduction in hybridization workload, each researcher can dedicate the saved time and energy to new variety hybridization work, allowing each person to conduct at least four more new variety hybridization projects. This not only improves the efficiency of researchers but also opens up a new path for high-throughput and precise soybean backcross breeding, providing a practical and valuable solution reference. It is expected to trigger a technological revolution in the field of soybean breeding, propelling soybean new variety breeding towards a more efficient and precise direction, laying a solid technological foundation for ensuring national food security and enhancing the competitiveness of the soybean industry.

[0202] (3) SSR marker identification

[0203] DNA was extracted from samples using a magnetic bead DNA extraction kit (Kangwei Century CW2361S). Extracted DNA was quality checked and concentration adjusted using a NanoDrop Eight spectrophotometer. A 5 μL reaction mixture was prepared per well (Table 9). Single-label amplification was performed using primers 38 pairs PG01-PG38 (National Variety Approval Test Purity Detection Primer NY / T2595-2025, Soybean Variety Authenticity Identification SSR Molecular Marker Method). Individual plants identical to the recurrent parent or with differences of less than 2 loci were selected from hybrid progeny or backcross progeny for subsequent experiments.

[0204] Table 9 Forgene reaction system (5μL reaction system)

[0205]

[0206] On a VeritiPro™ PCR instrument, the reaction program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 10 s, 60°C annealing extension for 25 s, 32 cycles; 72°C extension for 10 min.

[0207] Mix the PCR products according to the panel combination, and dilute them 400 times with ddH2O. Take 1.2 μl of the diluted PCR product and add it to 9 μl of Hi-Di 3730xl DNA analyzer containing Liz500.

[0208] The size of the fluorescently labeled DNA fragments obtained by PCR amplification was analyzed using GeneMapper.

[0209] Example 4

[0210] Self-crossing homozygotes

[0211] Based on the self-pollination and backcrossing directional breeding screening in Examples 1-3, plants that achieved a background recovery rate meeting the variety approval standards and also exhibited glyphosate tolerance were further subjected to homozygous self-pollination, as detailed below:

[0212] After achieving the required backcross background, we faced a key challenge: how to accelerate the multi-generation homozygous process. This invention cleverly modifies the key environmental factor of photoperiod, precisely regulating the growth rate of soybeans to achieve the goal of 4 to 5 generations of homozygosity per year.

[0213] To further investigate the specific impact of photoperiod on soybean growth and development and determine the optimal light conditions, this invention conducted a rapid propagation experiment in Hainan. During the experiment, different combinations of light duration were used for planting observation, and the photoperiod data accumulated in the previous hybridization platform was comprehensively analyzed. The study found (Table 10) that timely shading during the critical growth stage of true leaf unfolding (normal light duration in Hainan is 14 hours), with a light duration of 11 hours and a darkness duration of 13 hours, allowed for a relatively stable control of the soybean flowering period at approximately 22-28 days, which is a suitable light treatment method. Under normal conditions, the flowering period for materials from Northeast China is 30-35 days; for materials from the Huanghuai and Northwest regions, it is 32-37 days; and for materials from the South, it is approximately 40-43 days. Therefore, this method shortened the flowering period.

[0214] Table 10. Shortest flowering period for 13 varieties under 11h light conditions

[0215]

[0216] In summary, this study conducted a systematic and in-depth hybridization study using 315 soybean varieties (lines) as recurrent (recipient) parents and the glyphosate-tolerant transgenic soybean Zhonghuang 6106 as the donor parent. During the hybridization process, a carefully constructed soybean hybridization technology system was fully utilized, integrating advanced hybridization methods with precise environmental control measures to improve the success rate and efficiency of hybridization. Simultaneously, targeted backcross screening was performed on certain specific varieties using directional screening technology.

[0217] Through a series of rigorous and complex experimental procedures and technical treatments, fruitful results were achieved. In backcrossing, 183 varieties were successfully backcrossed once or more, with 85 varieties backcrossed twice or more, 62 varieties backcrossed three times or more, 30 varieties backcrossed four times or more, and even 12 varieties backcrossed five times or more. This abundant backcross data provides a solid foundation for further in-depth research on the genetic characteristics and variety improvement of soybeans.

[0218] Subsequently, advanced genotyping techniques were used to comprehensively test and analyze the backcrossed varieties. The results showed that 24 varieties achieved a genetic background restoration rate comparable to the recipient varieties, of which 12 were nationally approved varieties. Geographically, varieties from Northeast China performed best, with 16 varieties meeting this standard, followed by the Yellow River and Huai River regions with 5, Northwest China with 1, and South China with 2 (Table 11). This result not only reflects the differences in genetic stability during backcrossing among varieties from different regions but also provides important clues for further research on regional adaptability.

[0219] Table 11 List of recurrent parental genetic background recovery rates that meet national approval standards

[0220]

[0221] The main research results of this invention are as follows:

[0222] 1. High-efficiency hybrid soybean technology system

[0223] (1) High-efficiency soybean hybridization technology in Beijing: By comparing the effects of different single-factor treatments such as photoperiod, temperature, fertilizer and water treatment and hybridization method on the survival rate of hybrid fruits, the optimal treatment of each factor was selected and combined to establish a two-times-a-year soybean hybridization technology in Beijing. In the summer of Beijing (April to November), hybridization of 171 Northeast varieties, 45 Huang-Huai-Hai varieties, 49 Northwest varieties (lines) and 52 Southern varieties (lines) was completed.

[0224] (2) High-efficiency hybridization technology for soybeans in Hainan: By comparing the effects of different single-factor treatments such as photoperiod, temperature and hybridization method on the survival rate of hybrid fruits, a double-hybridization technology for soybeans in Hainan was established. This technology has overcome the problem of low survival rate of hybrid fruits under short-day conditions in Hainan and has completed the hybridization combination configuration of 112 Northeast varieties, 45 Huang-Huai-Hai varieties, 30 Northwest varieties and 42 Southern varieties.

[0225] By integrating and innovating the effects of light source, light duration, temperature, water and fertilizer management, pest and disease control, and stigma moisturizing after pollination on hybridization technology, a highly efficient hybridization technology system with four cycles per year (twice in Beijing and twice in Hainan) was developed. In Beijing, the survival rate of hybrid fruits remained stable at over 60%, with the highest being over 80% for the Zhongpin 661 variety. In Hainan, under short-day conditions, the survival rate of hybrid fruits increased from less than 2% to over 50%.

[0226] 2. Targeted backcross screening system

[0227] (1) Phenotypic selection: Foreground selection utilizes herbicide tolerance characteristics, and by spraying glyphosate, about 30% of false hybrids and non-resistant offspring can be eliminated; background selection uses traits such as flower color and pubescence to screen, and in the field, 50% of plants with traits highly similar to backcross parents can be selected. Combining target traits and genetic background with phenotypic selection, more than 70% of offspring can be eliminated, improving the efficiency of phenotypic selection.

[0228] (2) Genotypic directional selection: The KASP marker for the GAT gene was developed to improve the selection efficiency of glyphosate herbicide tolerance characteristics. Compared with phenotypic identification, the identification time can be saved by 13-20 days. Background detection was carried out using Zhongdouxin No. 1. The 5-7 plants with the highest recovery rate in each generation were selected for directional backcrossing. After 4 backcrosses, a background recovery rate of >97% was obtained. The SSR marker was detected using the national approval standard. Individuals with SSR <2 were screened to meet the new germplasm that met the approval standard for backcross varieties. This reduced the number of backcrosses by 2 and the number of hybridizations by 10 times compared with theoretical simulation.

[0229] (3) Rapid selection technology system for hybrid offspring: Herbicide spraying, the developed GAT gene KASP marker, and Zhongdouxin No. 1 were used to rapidly detect target traits and genetic background. Through the organic combination of phenotypic and genotypic identification, the workload of theoretical backcrossing 6 times and 50 plants per generation was reduced by 10 times. The cost of backcrossing and breeding of each variety was reduced by more than 40,000 yuan. The work of conventional 6 years was completed in 2 years, the personnel input was reduced by 90%, and the survival rate of hybrid fruits exceeded 50%, which significantly improved the efficiency of backcross breeding.

[0230] 3. Creation of new herbicide-tolerant germplasm

[0231] Utilizing efficient hybridization technology and a directional backcross screening system, 315 soybean varieties (lines) were hybridized, resulting in 183 varieties (BC1), 85 varieties (BC2), 62 varieties (BC3), 30 varieties (BC4), and 12 varieties (BC5). Twenty-four lines with high glyphosate tolerance and genetic background recovery rates meeting the national variety approval standards for backcrossing and conversion (SSR locus difference ≤1) were selected. This provides solid material support for the breeding of herbicide-tolerant transgenic soybean varieties.

[0232] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0233] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapid directional backcrossing and breeding of soybeans, characterized in that, Includes the following steps: (1) Selection of donor and recipient parents: Soybean varieties with glyphosate tolerance were selected as donor parents, and soybean varieties with excellent traits from the southern region, the Huang-Huai region, the northeast region and / or the northwest region were selected as recipient parents; (2) Hybridization: The donor parent and the recipient parent are hybridized, and hybrid grains are harvested; (3) F1 generation screening: The hybrid seeds harvested in step (2) are sown, and resistance screening, agronomic trait screening, and background detection are carried out in sequence using KASP molecular markers, agronomic trait phenotypes, and Zhongdouxin No. 1 chip. Plants with the same or similar agronomic traits as the recipient parent, high background recovery rate, high similarity to the recurrent parent, and glyphosate tolerance are selected for use. (4) Backcrossing: The plants selected in step (3) are backcrossed with the recipient parent, and BC1F0 is harvested; (5) BC1F1 generation screening: Sow the BC1F0 harvested in step (4), and combine KASP molecular markers, agronomic phenotypic and Zhongdouxin No. 1 chip to carry out resistance screening, agronomic phenotypic screening and background detection in sequence. In BC1F1, select 5-7 plants with agronomic traits that are the same or similar to the recipient parent, high background recovery rate, high similarity to the recurrent parent and glyphosate tolerance, and keep them for later use. (6) Repeated backcrossing: The plants obtained in step (5) are backcrossed with the recipient parent multiple times. The screening process of BC1F1 generation is repeated for each backcross generation. 5-7 plants from the backcross generation are selected for the next backcross. Through multiple backcrossings, when the SNP detection similarity rate between the parent and offspring of Zhongdouxin No. 1 is >97%, the SSR marker detection is carried out to meet the requirements of variety approval. Plants that meet the requirements of the number of differences in backcross SSR markers in the "National Transgenic Soybean Variety Approval Standard (Trial)" and also have glyphosate tolerance are obtained. (7) Self-pollination homozygosity: The plants obtained in step (6) are self-pollinated homozygosity. The self-pollinated offspring are subjected to photoperiod regulation of 11h light / 13h dark from the time the soybean true leaves unfold. When the plants enter the grain-filling stage, natural light and longitude management can be carried out to accelerate generation homozygosity and obtain new germplasm. The recurrent parents mentioned in steps (3) and (5) are the recipient parents. Step (2) When the hybridization location is Beijing, the specific hybridization method is as follows: A: Sowing: Sowing the donor parent and the recipient parent from different ecological zones in the Beijing area; B: Photoperiod Adjustment: Photoperiod adjustment was performed on parental varieties from different ecological zones. The photoperiod was adjusted to 8 hours of light / 16 hours of darkness for soybean varieties in southern regions; 12 hours of light / 12 hours of darkness for soybean varieties in the Huang-Huai region; 16 hours of light / 8 hours of darkness for soybean varieties in Northeast China; and 12 hours of light / 12 hours of darkness for soybean varieties in Northwest China. This shortened and homogenized the flowering period while extending the flowering duration. The flowering period refers to the period from seedling emergence to initial flowering; the flowering duration refers to the period from initial flowering to pod formation. C: Temperature compensation measures: After soybeans are sown in April or May each year, mulching is carried out. When the outdoor temperature is ≥20℃, mulching is stopped. D: Integrated water and fertilizer management: Step A: Apply slow-release compound fertilizer as base fertilizer to the soil at the same time as soybean sowing. From the V3 stage of soybean, apply urea, amino acids, trace elements and growth regulators as top dressing; routine water management. E: Hybridization technique: Males are removed and pollinated on the same day. After pollination, leaves are wrapped immediately. Hybridization is completed. The leaves are removed 2-5 days after hybridization. The lateral flower buds near the pods are removed 5-9 days after hybridization. The lateral flower buds near the pods are removed a second time 15-20 days after hybridization.

2. The method for rapid directional backcrossing and breeding of soybeans according to claim 1, characterized in that, In step E, the time for male removal is from 5:00 AM to 9:30 AM, and the time for pollination is from 9:30 AM to 11:30 AM.

3. The method for rapid directional backcrossing and breeding of soybeans according to claim 1, characterized in that, The KASP molecular marker reaction system described in step (3) is as follows: 10 μL reaction system, 5 μL 2×Master Mix, 0.01 μL 100 μM forward primer 1, 0.01 μL 100 μM forward primer 2, 0.03 μL 100 μM reverse primer, 25-250 ng DNA template, and ddH2O to bring the total to 10 μL; The KASP molecular marker reaction procedure in step (3) is as follows: 60℃ for 30 s; 95℃ for 10 min; 95℃ for 20 s, 61℃-55℃, -0.6℃ per cycle, annealing extension for 40 s, 10 cycles; 95℃ for 20 s, 55℃ annealing extension for 40 s, 30 cycles; then read the 60℃ for 30 s reading and perform KASP typing detection.

4. The method for rapid directional backcrossing and breeding of soybeans according to claim 1, characterized in that, The agronomic traits mentioned in step (3) are specific phenotypic traits of the recipient parent.

5. The method for rapid directional backcrossing and breeding of soybeans according to claim 1, characterized in that, The reaction system for the SSR molecular marker described in step (6) is as follows: 5 μL reaction system, 2.5 μL 2× PCR Mix, 0.1 μL 10 μM forward primer, 0.1 μL 10 μM reverse primer, 15-150 ng DNA template, and ddH2O to make up to 5 μL; The reaction procedure for the SSR molecular marker is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 25 s, 32 cycles; extension at 72℃ for 10 min. The number of SSR marker differences in backcrossing and breeding as specified in the "National Standards for the Approval of Genetically Modified Soybean Varieties (Trial)" in step (6) needs to be <2.

6. A method for rapid directional backcrossing and breeding of soybeans, characterized in that, Includes the following steps: (1) Selection of donor and recipient parents: Soybean varieties with glyphosate tolerance were selected as donor parents, and soybean varieties with excellent traits from the southern region, the Huang-Huai region, the northeast region and / or the northwest region were selected as recipient parents; (2) Hybridization: The donor parent and the recipient parent are hybridized, and hybrid grains are harvested; (3) F1 generation screening: The hybrid seeds harvested in step (2) are sown, and resistance screening, agronomic trait screening, and background detection are carried out in sequence using KASP molecular markers, agronomic trait phenotypes, and Zhongdouxin No. 1 chip. Plants with the same or similar agronomic traits as the recipient parent, high background recovery rate, high similarity to the recurrent parent, and glyphosate tolerance are selected for use. (4) Backcrossing: The plants selected in step (3) are backcrossed with the recipient parent, and BC1F0 is harvested; (5) BC1F1 generation screening: Sow the BC1F0 harvested in step (4), and combine KASP molecular markers, agronomic phenotypic and Zhongdouxin No. 1 chip to carry out resistance screening, agronomic phenotypic screening and background detection in sequence. In BC1F1, select 5-7 plants with agronomic traits that are the same or similar to the recipient parent, high background recovery rate, high similarity to the recurrent parent and glyphosate tolerance, and keep them for later use. (6) Repeated backcrossing: The plants obtained in step (5) are backcrossed with the recipient parent multiple times. The screening process of BC1F1 generation is repeated for each backcross generation. 5-7 plants from the backcross generation are selected for the next backcross. Through multiple backcrossings, when the SNP detection similarity rate between the parent and offspring of Zhongdouxin No. 1 is >97%, the SSR marker detection is carried out to meet the requirements of variety approval. Plants that meet the requirements of the number of differences in backcross SSR markers in the "National Transgenic Soybean Variety Approval Standard (Trial)" and also have glyphosate tolerance are obtained. (7) Self-pollination homozygosity: The plants obtained in step (6) are self-pollinated homozygosity. The self-pollinated offspring are subjected to photoperiod regulation of 11h light / 13h dark from the time the soybean true leaves unfold. When the plants enter the grain-filling stage, natural light and longitude management can be carried out to accelerate generation homozygosity and obtain new germplasm. The recurrent parents mentioned in steps (3) and (5) are the recipient parents. Step (2) When the hybridization location is Hainan, the specific hybridization method is as follows: A: Hybrid parent sowing: Sowing donor parents and recipient parents from different ecological zones in Hainan Province; B: Light compensation measures: Start light compensation when the soybean true leaves unfold and stop light compensation when the flower buds differentiate, then manage the natural light length. C: Temperature compensation measures: When soybeans begin to emerge and the air temperature is below 20℃, build a heat preservation shed to keep the temperature ≥20℃ throughout the entire growth stage of the plants. D: Integrated water and fertilizer management: Step A: Apply slow-release compound fertilizer as base fertilizer to the soil at the same time as soybean sowing. From the V3 stage of soybean, apply urea, amino acids, trace elements and growth regulators as top dressing; routine water management. E: Hybridization Technique: Parental hybridization is carried out between 9:00 and 15:

00. The specific hybridization method is as follows: pollination is carried out at the same time as emasculation. At the moment of completion of pollination, the leaf wrapping operation is performed. Hybridization is completed. The leaf wrapping is removed 2-5 days after the completion of hybridization. The lateral flower buds near the pod swelling flower are removed 5-9 days later. The lateral flower buds near the pod swelling flower are removed a second time 15-20 days later. In step B, the light compensation uses a light source with a specific light quality, whose spectral structure is as follows: 25% far-red light, 42% red light, 18% green light and 15% blue light. The wavelength range of the far-red light is 730-750nm; the wavelength range of the red light is 640-680nm; the wavelength range of the green light is 530-560nm; and the wavelength range of the blue light is 460-490nm. In step B, when the parent plant's ecological zone is in the south, the optimal light duration is 14-16 hours of light / 10-8 hours of darkness; when the parent plant's ecological zone is in the Yellow River and Huai River region, the optimal light duration is 16-18 hours of light / 8-6 hours of darkness; when the parent plant's ecological zone is in the northeast, the optimal light duration is 18-22 hours of light / 6-2 hours of darkness; and when the parent plant's ecological zone is in the northwest, the optimal light duration is 16-18 hours of light / 8-6 hours of darkness.

7. The method for rapid directional backcrossing and breeding of soybeans according to claim 6, characterized in that, The KASP molecular marker reaction system described in step (3) is as follows: 10 μL reaction system, 5 μL 2× Master Mix, 10.01 μL 100 μM forward primer, 0.01 μL 100 μM forward primer 2, 0.03 μL 100 μM reverse primer, 25-250 ng DNA template, and ddH2O to bring the total to 10 μL; The KASP molecular marker reaction procedure in step (3) is as follows: 60℃ for 30 s; 95℃ for 10 min; 95℃ for 20 s, 61℃-55℃, -0.6℃ per cycle, annealing extension for 40 s, 10 cycles; 95℃ for 20 s, 55℃ annealing extension for 40 s, 30 cycles; then read the 60℃ for 30 s reading and perform KASP typing detection.

8. The method for rapid directional backcrossing and breeding of soybeans according to claim 6, characterized in that, The agronomic traits mentioned in step (3) are specific phenotypic traits of the recipient parent.

9. A method for rapid directional backcrossing and breeding of soybeans according to claim 6, characterized in that, The reaction system for the SSR molecular marker described in step (6) is as follows: 5 μL reaction system, 2.5 μL 2× PCR Mix, 0.1 μL 10 μM forward primer, 0.1 μL 10 μM reverse primer, 15-150 ng DNA template, and ddH2O to make up to 5 μL; The reaction procedure for the SSR molecular marker is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 25 s, 32 cycles; extension at 72℃ for 10 min. The number of SSR marker differences in backcrossing and breeding as specified in the "National Standards for the Approval of Genetically Modified Soybean Varieties (Trial)" in step (6) needs to be <2.

10. The application of the rapid directional backcrossing and conversion method for soybean as described in any one of claims 1-9 in the breeding of new soybean germplasm.

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