High-throughput ByF1 breeding method based on male sterility

Abstract

The invention discloses a high-throughput ByF1 breeding method based on male sterility, and belongs to the technical field of plant hybridization. According to the method, the male sterile line and the restoring line are subjected to mixed sowing and natural pollination, so that rapid creation of million-level F1 combinations can be realized, and about 6000 combinations can be created within one year through only one sterile line, which is equivalent to the sum of annual workloads of 6-15 traditional breeding subjects. In the screened excellent single plants, the combination of 29 dominant parents can be accurately traced through comparison of the whole genome SNP loci; the hybrid wheat variety Jiumai 01 cultivated by the applicant is just one of the 29 dominant parent combinations. The yield advantage of the male parent Shannong 22 of the spring vein 01 planted in the Shahe Sing base of Jinan through testing for many years can reach 11%-31.8%, the feasibility of the method in practice is fully verified, the breeding efficiency of the strong advantage combination of wheat is greatly improved, and technical support is provided for accelerating breeding of hybrid wheat and conventional wheat varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant hybridization technology, and particularly relates to a high-throughput ByF1 breeding method based on male sterility. BACKGROUND

[0002] Wheat is an important food crop in the world, and about one-third of the world's population relies on it as their main food source. With the rapid growth of the population and the reduction of arable land, increasing the yield of wheat is the key to meeting future food demand. Although conventional wheat breeding has made great progress, the annual compound growth rate of new varieties is only 0.51%, and the yield improvement is slow. Compared with conventional varieties with the highest yield, hybrid wheat varieties have an average yield advantage of 136.5 kg / mu (10-20%), and the adaptability of hybrid wheat varieties is also higher than that of conventional wheat varieties. One of the main reasons for the lack of large-scale industrialization of wheat heterosis utilization is the lack of strong advantage hybrid combinations.

[0003] The lack of strong advantage hybrid combinations is mainly related to the characteristics of wheat itself. Because wheat is a self-pollinating crop, the success rate of cross-pollination in nature is very low. To date, artificial sexual hybridization is still the most commonly used method of wheat breeding, and the basic process is as follows: artificial sexual hybridization of wheat is carried out, and the obtained grains are hybrid seeds. These seeds are planted in the field, and F1 generation seeds are harvested. After one generation of planting, the F2 generation plants are obtained, which show a large degree of separation. Then, the plants are evaluated for plant and ear traits, disease resistance, stress resistance, etc. The offspring that meet the breeding target requirements are selected. After F5 generation, the traits are basically stable, and the plants can be released. Then, yield comparison, regional test, and other tests are carried out, and finally, the plants are approved for large-area planting. Therefore, from the beginning of wheat hybridization to the birth of a new high-yield and high-quality variety, the whole process often takes a long time of ten years. In order to obtain a large number of hybrid wheat strong advantage combinations, a large number of hybrid combinations need to be prepared and yield identification needs to be carried out for many years. This breeding method is not only time-consuming and labor-intensive, but also has low efficiency, and few strong advantage combinations are discovered, which affects the further development of the wheat breeding industry. In addition, the acquisition of wheat strong advantage parent combinations not only provides an important support for the breeding of excellent hybrid wheat, but also provides an important support for the breeding of excellent conventional wheat. Therefore, improving the breeding efficiency and discovering more strong advantage combinations of hybrid wheat are the urgent tasks currently faced by the wheat breeding industry. SUMMARY

[0004] To address the aforementioned limitations of existing technologies, the purpose of this invention is to provide a high-throughput ByF1 breeding method based on male sterility. We have developed a high-throughput ByF1 (Breeding your F1) breeding technology based on male sterility. This technology enables massive (e.g., millions) combinations of wheat parents, and through field screening and identification, as well as liquid-phase chip SNP locus detection, it can significantly improve the breeding efficiency of strong-performing wheat parent combinations, providing technical support for accelerating the breeding of hybrid and conventional wheat varieties.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a high-throughput ByF1 breeding method based on male sterility, comprising the following steps: (1) Construct a genotype database of sterile lines and restorer lines; (2) Hybridize the sterile line and restorer line to produce F1 combinations; (3) Conduct planting trials on the harvested F1 combinations; select and identify the final F1 individual plants, and infer their sterile lines and restorer lines based on the F1 liquid phase chip detection results to obtain strong-advantage combinations; (4) Hybridize the sterile line and restorer line obtained in step (3) to produce seeds.

[0006] Furthermore, in step (1), the sterile line carries a recessive nuclear sterility gene or a dominant nuclear sterility gene.

[0007] Furthermore, the recessive nuclear sterility gene is ms1 or Msg47 The dominant nuclear sterility gene mentioned is Ms2 .

[0008] Furthermore, the method for constructing a genotype database of sterile lines and restorer lines is as follows: Leaves of male-sterile lines and restorer lines with different genotype backgrounds were collected at the heading stage to extract DNA. SNP locus genotype data of male-sterile lines and restorer lines were collected, and a genotype database of male-sterile lines and restorer lines was successfully constructed.

[0009] Furthermore, in step (2), maintainer lines with different genotype backgrounds are sown and propagated, and each genotype maintainer line is harvested separately and a large number of sterile line seeds are obtained through color sorting; restorer lines with different genotype backgrounds are sown and propagated, and each restorer line is planted in one row with a row length of 1-1.5 meters and a row spacing of 20-30 centimeters. The sterile and restorer lines need to undergo at least three rounds of impurity removal during propagation to ensure seed purity.

[0010] Furthermore, in step (2), the number of grains used for each genotype of sterile line is not less than the number of restorer line genotypes; After sowing, carry out normal field management, watering, and prevention of weeds and pests; F1 combination consists of seeds harvested from a single sterile plant.

[0011] Furthermore, in step (3), normal field management, watering, and prevention of weeds and pests are carried out after sowing, but disease control is not required; The criteria for identifying and selecting F1 individual plants are as follows: (1) The number of ears is not less than 10; (2) The number of grains per ear is not less than 40; (3) The weight of 1,000 grains is not less than 40 grams; (4) The plant height is not higher than the local control variety; (5) The disease resistance is above medium resistance; (6) The cold resistance is stronger than the local conventional control variety.

[0012] Furthermore, in step (3), the method for F1 liquid phase chip detection is as follows: 20 self-pollinated seeds from the selected F1 single plant are bagged and germinated. When the seedling grows one leaf, the leaves are taken into the same sample tube to make a mixed sample and DNA is extracted. F1 liquid phase chip detection is performed targeting the SNP site.

[0013] Furthermore, the strong-advantage combinations in step (3) are used in hybrid wheat or conventional wheat breeding.

[0014] Furthermore, in step (4), the sterile line and the restorer line are hybridized to produce seeds, and the obtained hybrid wheat seeds are sown to identify the strong-advantage combination F1. The criteria for the F1 strong combination are as follows: Community area 7.5m² 2 The sowing density is 50,000 to 200,000 basic seedlings, and the yield of hybrid wheat is ≥8% higher than that of local conventional wheat control varieties.

[0015] The beneficial effects of this invention are: This invention first formulates a massive number of hybrid combinations, then conducts multi-environmental identification and screening, and finally accurately estimates the parents of the strong-performing combinations through liquid chromatography-mass spectrometry (LC-MS) detection. This invention is the first in the world to utilize F1 single plants for high-throughput breeding, achieving massive hybridization based on male sterility and determining parentage based on genome-wide SNP loci. This invention can achieve massive-scale hybridization of wheat parental combinations, and the field screening and identification, along with LC-MS SNP locus detection, can greatly improve the breeding efficiency of strong-performing wheat parental combinations, providing technical support for accelerating the breeding of hybrid and conventional wheat varieties.

[0016] This invention enables the rapid creation of millions of F1 hybrids by sowing a mixture of male-sterile lines and restorer lines and relying on natural pollination. In traditional methods, a single wheat breeding entity can create 400-1000 hybrids annually. However, in Example 3, this method created 6000 hybrids within a year using only one male-sterile line, equivalent to the combined annual workload of 6-15 traditional breeding entities. As the number of male-sterile lines increases, the number of hybrids will multiply, fundamentally overcoming the bottlenecks of traditional artificial hybridization in terms of scale, labor costs, and time efficiency. Combined with whole-genome SNP fingerprinting analysis technology, this method not only efficiently constructs ultra-large-scale hybrid populations but also allows for precise parentage tracing of each selected F1 dominant plant, ensuring the traceability of hybrid hybrids. Among the selected superior individual plants, the F1 individual plant 1 selected in Examples 3 and 4 (see Tables 3 and 4 for details) was accurately traced back to the hybrid wheat variety Quanmai 01, which was independently bred by the applicant, through whole-genome SNP locus comparison. After years of testing and planting at the Jinan Shahe Xin Base, its yield advantage over its parent Shannong 22 (number svg6684 or svg3894) can reach 11%-31.8%, which fully verifies the feasibility of the method in practice.

[0017] This invention addresses the core pain points of traditional breeding methods, such as cumbersome manual operations, time-consuming and labor-intensive processes, and the susceptibility to errors, mismatches, or missing records. It achieves high-throughput, high-efficiency, digitalized, and precise breeding. This not only promotes the transformation and upgrading of hybrid wheat breeding in my country from "experience-oriented" to "data-driven," but also possesses significant strategic industrial value, helping to enhance my country's independent innovation capabilities and international competitiveness in the seed industry. With the continuous decline in genotyping costs, this technology is expected to achieve widespread application and universal promotion.

[0018] This invention's technical system possesses broad applicability and excellent extensibility. It can be used not only for the efficient screening of strong-advantage combinations in hybrid wheat but also extended to the accelerated breeding of conventional wheat varieties. By integrating marker-assisted selection with double haploid (DH) technology, it achieves rapid homozygosity in offspring, significantly shortening the breeding cycle. The method of this invention has cross-species application potential and can be extended to crop systems that rely on artificial pollination, such as maize, rice, and rapeseed, providing a universally applicable technical path for the precision, data-driven, and large-scale development of global hybrid breeding. Attached Figure Description

[0019] Figure 1 Phylogenetic trees of 95 major wheat varieties from different ecological regions across the country. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0022] The ecological zones in this invention are divided as follows: the northern wheat region of the Yellow River and Huai River, the southern wheat region of the Yellow River and Huai River, the northern winter wheat region, the southern winter wheat region, the winter-spring mixed wheat region, and the northern spring wheat region.

[0023] A maintainer line refers to a collected or self-created wheat blue monoculture, such as the Chinese 4E-ms system (https: / / doi.org / 10.2135 / cropsci2005.0029). Wheat blue monocultures can self-pollinate and produce seeds of two colors. Blue seeds have 43 chromosomes and are self-pollinating maintainers (which are also the wheat blue monocultures themselves); white or amber seeds have 42 chromosomes and are wheat sterile lines based on a recessive nuclear male sterility gene. A restorer line refers to normal male-fertile wheat germplasm that can provide pollen to a wheat sterile line, enabling the latter to produce hybrids that can self-pollinate and produce seeds, thus restoring fertility.

[0024] The method for constructing the genotype database of sterile lines and restorer lines is as follows: 1.1 Propagation of maintainer lines with different genotype backgrounds. Six-row mechanical sowing was performed using a plot seeder. Each genotype maintainer line was harvested separately and color-sorted to obtain a large quantity of corresponding sterile line seeds.

[0025] 1.2 Propagation of restorer lines with different genotypes. Sowing was carried out using a row seeder, with one row planted for each restorer line, the row length being 1.2 meters and the row spacing being 25 centimeters.

[0026] 1.3 Leaves of sterile lines and restorer lines with different genotype backgrounds were collected at the heading stage to extract DNA. SNP locus genotype data (fingerprint maps) of sterile lines and restorer lines were collected using liquid phase chip technology to construct a genotype database of sterile lines and restorer lines.

[0027] In steps (1.1) to (1.2), the sterile line and restorer line need to undergo at least three rounds of impurity removal during propagation to ensure seed purity. In step (1.3), each plant whose leaves are collected is labeled and harvested separately when mature.

[0028] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1: Utilizing a 5K whole-genome cGPS liquid-phase chip based on recessive nuclear sterility genes ms1 or Msg47 High-throughput "ByF1 breeding" technology Using 60K whole-genome cGPS liquid phase chip data, 95 wheat varieties distributed across different ecological regions in China were classified into paternal and maternal parent groups. Figure 1 The male parent group included 60 wheat varieties from different ecological regions across the country used as restorer lines, and the sterile line LD560 of the independently bred hybrid wheat variety 'Quanmai 01' was developed. ms1 or Msg47 The recessive nuclear male sterility gene was used as the male sterile line; the grains of the restorer line and the male sterile line were completely mixed and sown for random mating. Based on the 5K whole genome cGPS liquid phase chip detection data analysis, high-throughput design of ByF1 breeding was realized.

[0030] The specific steps are as follows: 1. Construction of genotype database and pooled testing Genotype data (fingerprint maps) of SNP loci in sterile and restorer lines were collected using liquid-phase chip technology to construct a genotype database of sterile and restorer lines.

[0031] Ten seeds were taken from each of the 60 varieties in the male parent group and mixed evenly to form a restorer line mixed pool (24 g); the sterile line LD560 (24 g) was weighed and mixed with the restorer line mixed pool (24 g) in equal amounts to obtain a large mixed seed pool (48 g).

[0032] 2. Hybrid seed production In the first autumn, all the mixed seed material was planted in the Shahexin base in Jinan, Shandong Province using a wheat seed planter, with a seed spacing of 15 cm and a row spacing of 20 cm. After sowing, normal management, watering, and prevention of weeds and pests were carried out in the field of the base. In the second summer, the sterile line (LD560) was harvested and the fruiting single plants were mixed and threshed to obtain F1 grains.

[0033] 3. Field identification tests and acquisition of plants with strong dominance in combination In the autumn of the second year, the harvested F1 grains were planted in the Shahexin base using a seeder (15 cm grain spacing, 20 cm row spacing). After sowing, normal management, watering, and weed and pest control were carried out in the field at the base, but disease control was not required. In the summer of the third year, individual plants were selected based on comprehensive phenotypic traits in the field (plant height, disease resistance, yellowing, etc.). The selected plants were bagged before pollen shedding until harvest after grain formation. Indoor seed testing (plant height, number of ears, thousand-grain weight, number of grains per ear) was used to make a final selection for individual plants. The final target individual plants were the strong-dominant combination plants. When selecting target individual plants, the selected target individual plants had the following traits: 1) number of ears not less than 10; 2) number of grains per ear not less than 40; 3) thousand-grain weight not less than 40 grams; 4) plant height not greater than the local control variety; 5) disease resistance above moderate resistance; 6) cold resistance stronger than the local conventional control variety.

[0034] 4. Genotyping of plants from dominant combinations Twenty seeds from the self-pollination progeny of the selected single plant were selected, and after germination treatment, when the seedlings had grown one leaf, the leaves were taken from each plant and mixed into a sample tube for DNA extraction. Data were detected using a 5K whole-genome cGPS liquid chromatography chip. Data analysis was performed using SNP locus data from the sterile line LD560, the restorer line, and the selected F1 plant to estimate the restorer line corresponding to the selected F1 plant (Table 1).

[0035] Table 1. Restorer lines corresponding to the selected F1 individual plants 5. Field identification of plants selected from strong-performing combinations The sterile line and restorer line corresponding to the selected F1 single plant from step 4 were crossbred to produce hybrid wheat seeds. These seeds were then sown using a small-plot seeder at a density of 50,000 or 100,000 basic seedlings, with a plot area of ​​7.5m². 2 The control variety was a local conventional wheat variety (planting density of 150,000 or 200,000).

[0036] The field evaluation of high-yield combinations was based on a yield increase of ≥8% compared to the local conventional wheat control varieties (planting density of 150,000 or 200,000).

[0037] Example 2: Utilizing a 5K whole-genome cGPS liquid-phase chip based on dominant nuclear sterility genes Ms2 High-throughput "ByF1 breeding" technology The dwarf material containing 78 genetic backgrounds (BC4F1, dominant nuclear male sterility gene) was used. Ms2 With dominant dwarf gene Rht- D1A dominant male-sterile line (maternal parent) was used, and 60 wheat varieties from different ecological regions across the country (identified by 60K whole-genome cGPS liquid chip detection) were used as restorer lines (paternal parents). These were randomly mated after complete mixing and sowing. High-throughput design for ByF1 breeding was achieved based on 5K whole-genome cGPS liquid chip detection data analysis. The specific procedures are as follows: 1. Construction of genotype database and pooled testing Genotype data (fingerprint maps) of SNP loci in dominant sterile lines and restorer lines were collected using liquid-phase chip technology to construct a genotype database of sterile and restorer lines.

[0038] Ten seeds from each of the dwarf and atrophic materials from 78 genetic backgrounds were mixed evenly to obtain a dominant male-sterile line pool (maternal line pool). Ten seeds from each of the 60 wheat varieties promoted from different ecological regions across the country were mixed evenly to obtain a restorer line pool (paternal line pool). The maternal and paternal line pools were then mixed evenly.

[0039] 2. Hybrid seed production In the first autumn, wheat was planted at the Shahe Xin base in Jinan, Shandong Province using a wheat seeder, with a grain spacing of 15 cm and a row spacing of 20 cm. After sowing, normal management, watering, and weed and pest control were carried out in the base's field. In the second summer, short-stemmed individual plants were harvested. Ms2 / ms2 Rht-D1 / rht-D1 F1 seeds were obtained by threshing individual plants together.

[0040] 3. Field identification tests and acquisition of plants with strong dominance in combination In the second autumn, F1 seeds were planted in the Shahexin base in Jinan, Shandong Province using a wheat seeder, with a seed spacing of 15 cm and a row spacing of 20 cm. After sowing, normal management, watering, and weed and pest control were carried out in the field at the base, but disease control was not required. In the third summer, based on the comprehensive phenotypic traits in the field (plant height, disease resistance, yellowing, etc.), fertile single plants with normal plant height were selected. The single ears of the selected plants were bagged before pollen shedding until harvest after grain formation. Indoor seed testing (plant height, number of ears, thousand-grain weight, number of grains per ear) was used to make a final selection for the single plants. The final target single plants were the strong-dominant combination plants. When selecting target single plants, the selected target single plants had the following traits: 1) number of ears not less than 10; 2) number of grains per ear not less than 40; 3) thousand-grain weight not less than 40 grams; 4) normal plant height (i.e., not dwarf) but not taller than the local control variety; 5) disease resistance above moderate resistance; 6) cold resistance stronger than the local conventional control variety.

[0041] 4. Genotyping of plants from dominant combinations Twenty seeds from the self-pollinated progeny of the selected single plant were selected, germinated, and when the seedlings had one leaf, the leaves were collected into the same sample tube to form a mixed sample and DNA was extracted. Data were detected using a 5K whole-genome cGPS liquid chromatography chip. Data analysis was performed using SNP locus data from 78 genetic backgrounds (maternal parent, dominant male-sterile line) of the dwarf abortion material, 60 wheat varieties (paternal parent, restorer line) from different ecological regions across the country, and the selected F1 single plant to estimate the dominant male-sterile line and restorer line corresponding to the selected F1 single plant (Table 2).

[0042] Table 2. Restorer lines corresponding to the selected F1 individual plants 5. Field identification of plants selected from strong-performing combinations The sterile line and restorer line corresponding to the selected F1 single plant from step 4 were crossbred to produce hybrid wheat seeds. These seeds were then sown using a small-plot seeder at a density of 50,000 or 100,000 basic seedlings, with a plot area of ​​7.5m². 2 The control variety was a local conventional wheat variety (planting density of 150,000 or 200,000).

[0043] The field evaluation of high-yield combinations was based on a yield increase of ≥8% compared to the local conventional wheat control varieties (planting density of 150,000 or 200,000).

[0044] Example 3: Utilizing a 5K whole-genome cGPS liquid-phase chip based on recessive nuclear sterility genes ms1 High-throughput "ByF1 breeding" technology The collected 6,000 germplasm resources were used as restorer lines and sterile lines such as LD560 (including...). ms1 or Msg47 By randomly mating seeds of a recessive nuclear male sterility gene (indicating a genetic predisposition to sterility), and analyzing 5K whole-genome cGPS liquid phase chip data, high-throughput design for ByF1 breeding was achieved. The specific procedures are as follows: 1. Construction of genotype database and pooled testing SNP locus data of the male-sterile line LD560 and 6000 germplasm resources used as restorer lines were collected using a 5K whole-genome cGPS liquid phase chip to construct a genotype database of male-sterile and restorer lines. 30 seeds from each of the 6000 germplasm resources were taken and mixed evenly to form a restorer line mixed pool (6 kg). 6 kg of the male-sterile line LD560 and 6 kg of the restorer line mixed pool were weighed and mixed in equal amounts to obtain a large mixed F1 seed pool (12 kg). To achieve low-density sowing, 88 kg of other common wheat seeds were inactivated and then mixed with the 12 kg of large mixed F1 seeds using a mixer to obtain 100 kg of seeds.

[0045] 2. Hybrid seed production In the first autumn, a wheat precision seeder was used to plant all the mixed material with inactivated seeds at a rate of 10 kg per mu (approximately 667 square meters). The actual sowing rate of F1 live seeds was 1.2 kg per mu, and a total of 10 mu were sown. After sowing, normal management, irrigation, and weed and pest control were carried out in the field at the base. In the second summer, the sterile line (LD560) was harvested and the fruiting individual plants were threshed to obtain F1 grains.

[0046] 3. Field identification tests and acquisition of plants with strong dominance in combination In the second autumn, the harvested F1 seeds were planted at 16 experimental sites in 16 different ecological zones across multiple provinces and regions in China (Shandong, Hebei, Henan, Jiangsu, Anhui, Shaanxi, Gansu, Chongqing, Sichuan, and Tibet). Each experimental site covered 3 mu (approximately 0.2 hectares), with a sowing rate of 10 kg per mu (1.5 kg of live F1 seeds and 8.5 kg of ordinary inactivated seeds). After sowing, normal management, watering, and weed and pest control were carried out in the field, but disease control was not required. In the third summer, individual plants were selected based on comprehensive phenotypic traits in the field (plant height, disease resistance, yellowing, etc.). The selected plants were bagged before pollen shedding and harvested after fruit set. The final selection of individual plants was conducted through indoor testing (plant height, number of ears, thousand-grain weight, number of grains per ear). The final target individual plants were the strong-viability combination plants. When selecting target plants, the selected target plants should have the following characteristics: 1) No less than 10 ears; 2) No less than 40 grains per ear; 3) No less than 40 grams per thousand grains; 4) Plant height no greater than the local control variety; 5) Disease resistance at or above the medium level; 6) Cold resistance stronger than the local conventional control variety.

[0047] 4. Genotyping of plants from dominant combinations Twenty seeds from the self-pollination progeny of the selected single plant were selected, and after germination treatment, when the seedlings had grown one leaf, the leaves were taken from each plant and mixed into a sample tube to extract DNA. Data were detected using a 5K whole-genome cGPS liquid chromatography chip. Data analysis was performed using SNP locus data from the sterile line LD560, the restorer line, and the selected F1 plant to estimate the restorer line corresponding to the selected F1 plant (Table 3).

[0048] Table 3. Restorer lines corresponding to the final F1 single plants 5. Field identification of plants selected from strong-performing combinations The sterile line and restorer line corresponding to the selected F1 single plant from step 4 were crossbred to produce hybrid wheat seeds. These seeds were then sown using a small-plot seeder at a density of 50,000 or 100,000 basic seedlings, with a plot area of ​​7.5m². 2 The control variety was a local conventional wheat variety (planting density of 150,000 or 200,000).

[0049] The field evaluation of high-yield combinations was based on a yield increase of ≥8% compared to the local conventional wheat control varieties (planting density of 150,000 or 200,000).

[0050] Example 4: Utilizing a 5K whole-genome cGPS liquid-phase chip based on recessive nuclear sterility genes ms1 or Msg47 High-throughput "ByF1 breeding" technology (single-mix) The collected 6,000 germplasm resources were used as restorer lines and sterile lines such as LD560 (including...). ms1 or Msg47 By modifying seed mixing and sowing methods using a recessive sterility gene, localized random mating between sterile and restorer lines can be achieved. High-throughput design for F1 breeding can be realized based on 5K whole-genome cGPS liquid phase chip detection data analysis. The specific steps are as follows: 1. Construction of genotype database and pooled testing SNP locus data of the sterile line LD560 and 6000 germplasm resources used as restorer lines were collected using a 5K whole-genome cGPS liquid phase chip to construct a genotype database of sterile and restorer lines. Thirty seeds from each of the 6000 germplasm resources were placed in seed bags, and 10 sterile seeds were mixed into each seed bag to obtain 6000 mixed materials of known restorer and sterile lines.

[0051] 2. Hybrid seed production In the first autumn, 6,000 single-harvest mixed materials were sown in 6-row plots using a wheat single-ear planter, with a row length of 1.2 meters and a row spacing of 20 centimeters. After sowing, normal management, watering, and prevention of weeds and pests were carried out in the field. In the second summer, the grains from the sterile line (LD560) were harvested row by row. F1 grain pools were obtained by mixing the grains in each 6-row plot, resulting in a total of 938 6-row mixed F1 grain pools, which were then labeled accordingly.

[0052] 3. Field identification tests and acquisition of plants with strong dominance in combination In the second autumn, the 938 F1 grain beds in 6-row plots were sown using a wheat seeder. The plots were 6 meters long, with a grain spacing of 15 centimeters and a row spacing of 20 centimeters. After sowing, normal management, watering, and weed and pest control were carried out in the field, but disease control was not required. In the third summer, individual plants were selected based on comprehensive phenotypic traits in the field (plant height, disease resistance, yellowing, etc.). The plot number corresponding to each individual plant was recorded, and the ears of the selected plants were bagged before pollen shedding until harvest after grain formation. The final selection of individual plants was conducted through indoor evaluation (plant height, number of ears, thousand-grain weight, number of grains per ear) (the plot number corresponding to each individual plant was recorded), and representative ears were bagged before pollen shedding and grain formation. The final target individual plants were the strong-dominant combination plants. When selecting target plants, the selected target plants should have the following characteristics: 1) No less than 10 ears; 2) No less than 40 grains per ear; 3) No less than 40 grams per thousand grains; 4) Plant height no greater than the local control variety; 5) Disease resistance at or above the medium level; 6) Cold resistance stronger than the local conventional control variety.

[0053] 4. Genotyping of plants from dominant combinations Twenty seeds from the self-pollination progeny of the selected single plant were selected, germinated, and when the seedlings had one leaf, the leaves were collected into the same sample tube to form a mixed sample and DNA was extracted. The DNA was detected using a 5K whole-genome cGPS liquid phase chip. Data analysis was performed using the SNP locus data of the sterile line LD560, restorer lines (six restorer lines corresponding to the plots of the single plant), and the selected F1 single plant to estimate the restorer line corresponding to the selected F1 single plant (Table 4).

[0054] Table 4. Restorer lines corresponding to the selected F1 individual plants 5. Field identification of plants selected from strong-performing combinations The sterile line and restorer line corresponding to the selected F1 single plant from step 4 were crossbred to produce hybrid wheat seeds. These seeds were then sown using a small-plot seeder at a density of 50,000 or 100,000 basic seedlings, with a plot area of ​​7.5m². 2 The control variety was a local conventional wheat variety (planting density of 150,000 or 200,000).

[0055] The field evaluation of high-yield combinations was based on a yield increase of ≥8% compared to the local conventional wheat control varieties (planting density of 150,000 or 200,000).

[0056] Example 5: Using a 5K whole-genome cGPS liquid-phase chip based on nuclear sterility recessive genes ms1 or Msg47 High-throughput "ByF1 breeding" technology The collected wheat varieties were classified according to their corresponding suitable ecological zones. Varieties from different ecological zones were used as restorer lines (paternal lines) and two independently developed nuclear male sterile lines, 23LD560 and 23LD272 (including...). ms1 or Msg47 By modifying seed mixing and sowing methods using a recessive sterility gene, random mating of sterile and restorer lines can be achieved. High-throughput design for ByF1 breeding is realized through data analysis using a 5K whole-genome cGPS liquid phase chip. The specific steps are as follows: 1. Construction of genotype database and pooled testing SNP locus data of sterile lines 23LD560 and 23LD272 and wheat varieties from different ecological regions used as restorer lines were collected using 5K whole-genome cGPS liquid phase chip detection to construct a genotype database of sterile and restorer lines. Sterile lines (maternal) 23LD560 and 23LD272 were mixed, and paternal lines from different ecological regions (about 20 per group) were mixed according to the same ecological region. Each group of materials was sown in plots, with 2 rows of sterile lines (20 seeds per row) and 4 rows of paternal lines per plot (5 paternal lines per row, 4 seeds per paternal line).

[0057] 2. Hybrid seed production In the first autumn, a seed drill was used to sow seeds in 6-row plots (plots / groups), with a row length of 2 meters, a plot spacing of 50 centimeters, and a grain spacing of 10 centimeters. Rows 2 and 5 were sterile lines, and rows 1, 3, 4, and 6 were male parent lines. After sowing, normal management, irrigation, and weed and pest control were carried out in the field. In the second summer, rows 2 and 5 of each plot were harvested, and F1 grains were mixed and threshed according to plot.

[0058] 3. Field identification tests and acquisition of plants with strong dominance in combination In the second autumn, the harvested F1 grains from different plots were sown in plots using a wheat seeder. The plot length was 6 meters, the grain spacing was 15 centimeters, and the row spacing was 20 centimeters. After sowing, normal management, watering, and weed and pest control were carried out in the field, but disease control was not required. In the third summer, individual plants were selected based on comprehensive phenotypic traits in the field (plant height, disease resistance, yellowing, etc.). The plot number corresponding to each individual plant was recorded, and the single ears of the selected plants were bagged before pollen shedding until harvest after grain formation. The final selection of individual plants was conducted through indoor seed evaluation (plant height, number of ears, thousand-grain weight, number of grains per ear) (the plot number corresponding to each individual plant was recorded), and representative single ears were bagged before pollen shedding and grain formation. The final target individual plants were the strong-dominant combination plants. When selecting target plants, the selected target plants should have the following characteristics: 1) No less than 10 ears; 2) No less than 40 grains per ear; 3) No less than 40 grams per thousand grains; 4) Plant height no greater than the local control variety; 5) Disease resistance at or above the medium level; 6) Cold resistance stronger than the local conventional control variety.

[0059] 4. Genotyping of plants from dominant combinations Twenty seeds from the self-pollinated progeny of the selected single plant were selected, germinated, and when the seedlings had one leaf, the leaves were collected into the same sample tube to form a mixed sample and DNA was extracted. The DNA was detected using a 5K whole-genome cGPS liquid chromatography chip. Data analysis was performed using SNP locus data from sterile lines 23LD560 and 23LD272, restorer lines (20 restorer lines corresponding to the plots of single plants), and the selected F1 single plant to estimate the restorer line corresponding to the selected F1 single plant (Table 5).

[0060] Table 5. Restorer lines corresponding to the final F1 individual plants 5. Field identification of plants selected from strong-performing combinations The sterile line and restorer line corresponding to the selected F1 single plant from step 4 were crossbred to produce hybrid wheat seeds. These seeds were then sown using a small-plot seeder at a density of 50,000 or 100,000 basic seedlings, with a plot area of ​​7.5m². 2 The control variety was a local conventional wheat variety (planting density of 150,000 or 200,000).

[0061] The field evaluation of high-yield combinations was based on a yield increase of ≥8% compared to the local conventional wheat control varieties (planting density of 150,000 or 200,000).

[0062] Example 6: Conventional breeding application of high-throughput "ByF1 breeding" technology based on nuclear male sterility gene High-throughput "ByF1 breeding" technology based on nuclear male sterility genes yielded strong heterotic F1 combinations (Examples 1-5). By combining this with molecular markers for nuclear male sterility genes, the F2 generation underwent screening and removal of the nuclear male sterility gene, rapidly obtaining homozygous fertile F2 generation individual plants. This enabled the rapid cultivation of conventional varieties using rapid breeding techniques (double haploid technology). Currently, recessive nuclear male sterility genes are being used... ms1 Let's take an example to illustrate: After the F2 seeds of the strong heterosis combination germinated, they were planted in 10×20 seedling trays. When the seedlings reached the one-leaf-one-heart stage, DNA was extracted from the leaves and analyzed using a recessive nuclear male sterility gene. ms1 Molecular markers were used for screening (Table 6). Based on the molecular marker detection results, genes not containing recessive nuclear sterility genes were screened. ms1 F2 seedlings were then vernalized and transplanted into an artificial climate chamber. After reaching the tasseling stage, artificial emasculation and corn pollination were performed. Haploid embryos were obtained through hormone treatment, and haploid seedlings were obtained through embryo rescue. Subsequently, chromosome doubling treatment with colchicine was used to obtain double haploid plants (pure lines). Finally, after field propagation and evaluation, superior wheat lines were obtained and entered the variety approval trial process.

[0063] Table 6. Genes used for nuclear sterility ms1 ms1 Detected molecular markers Results verified that conventional wheat lines created using strong hybrid vigor combinations exhibited excellent overall performance in the field, indicating that the high-throughput "ByF1 breeding" technology based on nuclear male sterility genes has accelerated the breeding of conventional wheat varieties.

[0064] This invention enables high-throughput hybrid combination F1, and through field screening and whole-genome SNP liquid-phase chip detection, it can greatly improve the breeding efficiency of strong wheat combinations, providing technical support for accelerating the breeding of hybrid wheat varieties and conventional wheat varieties.

[0065] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A high-throughput ByFl breeding method based on male sterility, characterized in that, It comprises the following steps: (1) constructing a genotypic database of the sterile line and the restorer line; (2) hybridizing the sterile line and the restorer line to breed seeds and planting, to obtain F1 combinations; (3) planting the harvested F1 combinations to perform a test; selecting and identifying a F1 single plant, and deducing the sterile line and the restorer line according to the F1 liquid chip detection result, to obtain a strong dominant combination; (4) hybridizing the sterile line and the restorer line of the strong dominant combination obtained in step (3) to breed seeds.

2. The male sterility based high-throughput ByF1 breeding method according to claim 1, characterized in that, In step (1), the sterile line carries a recessive nuclear sterile gene or a dominant nuclear sterile gene.

3. The high-throughput ByF1 breeding method based on male sterility according to claim 2, characterized in that, The recessive nuclear male sterile gene is ms1 or Msg47 The dominant nuclear male sterile gene is Ms2 .

4. The male sterility based high throughput ByFl breeding method according to claim 1, characterized in that, The method for constructing the genotypic database of the sterile line and the restorer line is as follows: At the heading stage, the sterile line and the restorer line with different genotypic backgrounds are collected for leaf collection, DNA extraction, collection of SNP site genotypic data of the sterile line and the restorer line, and successful construction of the genotypic database of the sterile line and the restorer line.

5. The male sterility based high-throughput ByFl breeding method according to claim 4, characterized in that, In step (2), the maintainer line with different genotypic backgrounds is sowed and expanded, and each genotypic maintainer line is separately harvested, and a large number of sterile line seeds are obtained through color selection; the restorer line with different genotypic backgrounds is sowed and expanded, and each restorer line is planted in one row with a length of 1-1.5 meters and a row spacing of 20-30 centimeters; The sterile line and the restorer line need to be at least 3 times of impurity removal during the expansion process to ensure the seed purity.

6. The male sterility based high throughput ByFl breeding method according to claim 1, characterized in that, In step (2), the use amount of the sterile line of each genotype is not less than the number of genotypes of the restorer line; Normal field management, watering and prevention of weeds and diseases are performed after sowing; The F1 combination is the seed harvested by the sterile line single plant.

7. The male sterility based high throughput ByFl breeding method according to claim 1, characterized in that, In step (3), normal field management, watering and prevention of weeds and diseases are performed after sowing, but disease control is not required; The standards for identifying and selecting the F1 single plant are as follows: (1) the number of ears is not less than 10; (2) the number of grains per ear is not less than 40; (3) the thousand-grain weight is not less than 40 grams; (4) the plant height is not higher than the local control variety; (5) the disease resistance is above medium resistance; (6) the cold resistance is stronger than the local conventional control variety.

8. The male sterility based high throughput ByFl breeding method according to claim 1, characterized in that, In step (3), the method for F1 liquid chip detection is as follows: 20 grains of the bagged self-pollination seeds of the selected F1 single plant are germinated, and when the seedlings grow 1 leaf, the leaves are collected into a mixed sample and DNA is extracted, and F1 liquid chip detection is performed on the SNP site.

9. The male sterility based high throughput ByFl breeding method according to claim 1, characterized in that, The strong dominant combination in step (3) is used for hybrid wheat or conventional wheat breeding.

10. The male sterility based high throughput ByFl breeding method according to claim 1, characterized in that, In step (4), the sterile line and the restorer line are hybridized to breed seeds, and the hybrid wheat seeds obtained are sowed to perform F1 identification of the strong dominant combination; The identification criteria of the strong advantage combination F1 are as follows: the plot area is 7.5 m 2 , the seeding density is 50,000-200,000 basic seedlings, and the hybrid wheat yield is increased by ≥8% compared with the local conventional wheat control variety.

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