A method for improving the yield of corn hybrid seed production by using a relative inbred line

Abstract

The application relates to a method for improving the seed yield of an excellent single-cross hybrid by using a close relative inbred line, which comprises the following steps: excellent single-cross hybrid selection, close relative inbred line collection and identification, close relative inbred line combining ability determination and analysis, complex female parent assembly, excellent single-cross hybrid improvement, parent and excellent single-cross hybrid evaluation, and seed yield comparison. The method adopts the idea that the seed yield of a corn three-cross hybrid can be improved, a complex female parent is assembled by using a close relative inbred line which has 1-3 site differences with the female parent of the excellent single-cross hybrid, the consistency, stability and specificity of the original single-cross hybrid are retained, and the plant of the improved complex female parent is robust. The method can improve the corn seed yield, has strong practicability and operability, is clear in purpose, can save cost, and can be popularized and applied in the corn seed production process.

Description

TECHNICAL FIELD

[0001] The application relates to a method for improving the seed production of corn single-cross by using a close relative inbred line, and belongs to the technical field of crop breeding. BACKGROUND

[0002] China is a large agricultural producer in the world, and the corn yield ranks among the top in the world. In 2007, the corn planting area in China exceeded the rice planting area. In 2012, the total corn yield exceeded the total rice yield, becoming the first grain crop in China. In 2020, the domestic corn yield was 261 million tons, accounting for 38.9% of the total grain yield. Among the three major grains of rice, wheat and corn, corn is widely distributed and has high yield, and is an important grain, economic and feed crop, and plays an important strategic role in ensuring food security.

[0003] In recent years, regional severe weather has occurred frequently, and plant diseases have been serious. The promotion and application of single-cross are greatly challenged. At the same time, a large number of new varieties are approved, the seed production is low, and the seed production cost in the northwest continues to increase, resulting in an increasing cost of new variety promotion year by year.

[0004] In view of the low seed production and high seed production cost of single-cross, a large number of researches have been made by predecessors, mainly including water and fertilizer effective utilization and corn plant type improvement. There are also researches on the improvement of seed production by three-cross breeding, but the uniformity and stability of three-cross corn are poor, and the consistency, stability and specificity of varieties are basic traits of varieties. The use of three-cross to improve seed production has not been popularized. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides a method for improving the seed production of corn single-cross by using a close relative inbred line. The method uses the idea that corn three-cross can improve the seed production, and uses a close relative inbred line having 1-3 site differences with an excellent single-cross female parent to form a composite female parent. While the consistency, stability and specificity of the original single-cross are retained, the improved composite female parent is robust and has high yield, thereby realizing the improvement of the seed production of the excellent single-cross.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] A method for improving the seed production of corn single-cross by using a close relative inbred line, taking an excellent single-cross female parent as the research object, screening a close relative inbred line having 1-3 molecular difference values with the excellent single-cross female parent through germplasm resource collection and screening, and using the close relative inbred line to form a composite female parent. Through the improvement of the excellent single-cross, a variety consistent with the composite female parent and the single-cross is selected. Then, the yield and yield component traits thereof are evaluated.

[0008] The method for improving the yield of maize single-cross seed production using closely related inbred lines includes the following steps:

[0009] (1) Selection of superior single-cross varieties: Select single-cross varieties with excellent traits such as high yield, wide adaptability, stress resistance, high quality and high resistance;

[0010] (2) Collection and identification of closely related inbred lines: Based on the superior single-cross maternal parent A1, the maternal parent was classified into groups using SSR molecular marker technology; germplasm resources of the same group as the superior single-cross maternal parent A1 were collected, and the specificity analysis between the collected germplasm resources and A1 was performed using SSR molecular marker technology to screen out closely related inbred lines that have 1-3 molecular differences from the superior single-cross maternal parent A1;

[0011] (3) Combining ability determination and analysis of closely related inbred lines: Using the superior single cross male parent B as the test species, and the collected closely related inbred lines and the superior single cross female parent A1 as the female parent, single crosses were bred. The combining ability of the bred single crosses was determined and analyzed, and closely related inbred lines (♀)×B(♂) and A1(♀)×B(♂) with the same high combining ability were screened out as closely related inbred lines A2.

[0012] (4) Combination of composite female parents: Sow seeds of A1 and A2, and obtain composite female parents A3 and A4 by orthogonal combination A2(♀)×A1(♂) and reciprocal combination A1(♀)×A2(♂);

[0013] (5) Improvement of superior single crosses: Sow homozygous female parents A1 and A2, composite female parents A3 and A4 and superior single cross male parent B. Use A1, A2, A3 and A4 as female parents and superior single cross male parent B as male parent to fertilize single crosses A1(♀)×B(♂), A2(♀)×B(♂), A3(♀)×B(♂), and A4(♀)×B(♂) to obtain single cross H1 and improved single crosses H2, H3 and H4 respectively.

[0014] (6) Evaluation of parents and superior single crosses: Four female parents A1, A2, A3, A4 and four single crosses H1, H2, H3, H4 were sown respectively. The consistency and specificity of the four female parents and four single crosses were analyzed using maize SSR molecular marker technology and DUS test technology.

[0015] (7) Seed production yield comparison: The yield and yield composition traits of the single crosses H1, H2, H3 and H4 obtained in step (5) were evaluated.

[0016] Among them, the superior single-cross female parent A1 is the inbred line JunM6968 of the JunDan58 female parent, A2 is the closely related inbred line HB37 of JunM6968, the superior single-cross male parent B is the paternal parent Jun262 of JunDan58, and H1 is the improved superior single-cross JunDan58.

[0017] The method for combining the composite female parent in step (4) is as follows: Sow 200-300 homozygous parent plants A1 and A2 respectively. During the pollen shedding and silking period, select 100 plants of A1 and A2 respectively for self-pollination to obtain homozygous female parents A1 and A2. Select 10-30 superior single plants with strong plants, coordinated male and female reproductive functions and large pollen production from A1, mix them evenly, and pollinate the remaining 100-200 single plants of A2 to obtain composite female parent A3. Select 10-30 superior single plants with pollen from A2, mix them evenly, and pollinate the remaining 100-200 single plants of A1 to obtain composite female parent A4.

[0018] The specific method for improving the superior single cross in step (5) is as follows: using homozygous maternal parents A1, A2, and composite maternal parents A3, A4 as maternal parents, and superior single cross B as paternal parents, sowing is carried out with a row ratio of maternal parent: paternal parent = 3:1. The male ears of the maternal parents are removed before the female parents tassel, and open isolation pollination is carried out to obtain the original single cross H1 and improved single cross H2, H3, and H4.

[0019] The evaluation method for the parents and superior single crosses in step (6) is as follows: 200-300 plants of the four maternal parents A1, A2, A3, A4 and the four single crosses H1, H2, H3, H4 are sown respectively. When the maize grows to the 3-leaf stage, 10 leaves from each material are mixed and molecular identification is performed using the 20 pairs of core SSR primers published in GB / T 39914-2021. After the emergence of A1, A2, A3, A4 and H1, H2, H3, H4, 40 plants are randomly selected from each area and marked. According to the plant variety specificity, uniformity and stability test guidelines published in GB / 19557.24-2018, 39 mandatory traits are selected from 60 maize varieties in the DUS test items to analyze and identify the uniformity and specificity of the four maternal parents and the four single crosses.

[0020] The specific method for comparing seed production yield in step (7) is as follows: during the corn seed maturity period in step (5), harvest 4 hybrid seeds, select 10 sample ears from each area, examine the yield composition and ear traits, compare and analyze with the original single cross, and select superior germplasm.

[0021] Beneficial effects of this invention:

[0022] 1. This application is based on the idea of ​​improving seed production yield through single-cross and double-cross hybrids. It utilizes superior single-cross female parents and closely related inbred lines to create composite female parents. Because the offspring of the composite female parents are robust and the parental yield is high, the seed production yield can be significantly increased, thereby reducing the seed production cost. At the same time, it uses closely related female parent inbred lines with differences of less than 4 gene loci to improve the hybrid parents. There are obvious differences and advantages between single-cross and triple-cross hybrids. The two hybrid female parents are closely related, and their offspring have highly consistent genes. The improved hybrids have the stability, uniformity, and high yield characteristics of superior single-cross hybrids. Seed production yield is increased without changing stability. This breaks through the traditional methods of increasing seed production yield, such as reciprocal cross seed production, cultivation technology improvement, and the use of modern mechanization technology, and achieves innovation in the method of increasing seed production yield.

[0023] 2. To ensure that new maize varieties possess a certain degree of independent innovation, the latest approval standards define varieties as having a molecular difference value ≥4. The new approval method has raised the standards for variety approval, leading to the obsolescence of some closely related inbred lines with superior traits, resulting in a waste of germplasm resources. This application, through analysis of existing germplasm resources, proposes a method to improve single-cross varieties using closely related inbred lines that differ from the superior single-cross parent by <4 loci, while maintaining the heterosis pattern, stress resistance, and adaptability of the inbred lines. This addresses the problem of low seed yield in single-cross varieties and effectively solves the waste of closely related single-cross varieties, germplasm resources, and genetic information, achieving an innovative and efficient utilization of germplasm resources.

[0024] 3. Currently, maize breeding in my country emphasizes variety innovation, combining modern bio-breeding techniques with traditional breeding methods. A large number of superior varieties have been introduced to the market. Many breeders still rely on the gradual infiltration of superior genes from inbred lines to improve germplasm. Building upon previous breeding experience, this application innovatively utilizes inbred lines with <4 gene loci differences to create composite maternal lines. This ensures the stability of parental offspring while improving superior single-cross varieties, providing a new strategy for improving maize breeding methods and enhancing superior single-cross varieties. Attached Figure Description

[0025] Fig. 1 Comparison of seedling traits between JunM6968 (A1) and closely related inbred lines A2 and composite parents (A3, A4);

[0026] Fig. 2 Comparison of seedling traits during silking and pollination period between Jun M6968 (A1) and closely related inbred lines A2 and composite parents (A3, A4);

[0027] Note: From left to right, they are: ♀-1, ♀-2, ♀-3, ♀-4;

[0028] Fig. 3 Comparison of ear traits between Jun M6968 (A1) and closely related inbred lines A2 and composite parents (A3, A4). Detailed Implementation

[0029] The specific implementation methods of this application will be further described in detail below with reference to the embodiments.

[0030] This application utilizes the inbred line JunM6968, a close relative of JunM6968, to improve the single-cross hybrid JunDan58 and increase seed production.

[0031] Example 1: Collection and identification of superior single-cross maternal inbred lines

[0032] 1. Selection of superior single-cross varieties

[0033] The maternal inbred line JunM6968 of the new maize variety Jundan 58, which has excellent traits such as high yield, wide adaptability, stress resistance, high quality, and high resistance, was selected as the base material. This variety is a superior single-cross hybrid with excellent traits, which is subject to restrictions on promotion and yield.

[0034] 2. Germplasm resource collection

[0035] In the summer of 2019, the inbred line JunM6968, the maternal parent of JunDan 58, with the pedigree of (PH6WC / PH09B / / PH6WC) / / Zheng58, was classified as a modified Reid group using SSR molecular markers. In the same year, 72 accessions of improved Reid group germplasm resources from superior single-cross maternal parents, created independently, were collected. 52 accessions of modified Reid group germplasm resources were also collected using platforms such as the National Maize Industry Technology System and the Henan Provincial Maize Genetic Improvement Academician Workstation.

[0036] 3. Germplasm resource screening

[0037] In 2019, based on the target superior single-cross hybrid maternal parent Jun M6968(A1), the maternal parent was classified using SSR molecular marker technology. Germplasm resources of the superior single-cross hybrid maternal parent Jun M6968 were collected, and then, using SSR molecular marker technology, the 124 collected materials were compared with 125 materials of Jun M6968 for specificity analysis. It was found that among the 124 materials, three closely related inbred lines, HB12, HB37, and HB52, which were created independently, differed from Jun M6968(A1) by 1-3 loci. Among them, HB12 (pedigree: PH6WC / Jun 658 / / Zheng 58); HB37 (pedigree: PH6WC / PH09B / / Zheng 58); and HB52 (pedigree: (PH6WC / / PH09B / Jun 658) / / Zheng 58).

[0038] The specific operational process of SSR molecular markers includes the following steps:

[0039] (1) DNA extraction and detection: When the corn reaches the 3-leaf stage, 10 leaves from each plot are mixed, ground into powder with liquid nitrogen, and DNA is extracted using the modified CTAB method. The quality and concentration of the extracted DNA are detected by 1.2% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer.

[0040] (2) SSR marker identification results analysis: The 20 pairs of core SSR primers published in GB / T 39914-2021 were used. PCR amplification was performed on the extracted DNA samples, following the procedures outlined in the Technical Specifications for Maize Variety Identification – SSR Marker Method. The amplification products were represented by "0" and "1" to indicate the presence or absence of the amplified fragment, and the allele types of each maize variety were recorded. The number of differentially expressed sites between each pair of samples was counted; this number represents the molecular difference between the two samples.

[0041] This application utilizes closely related inbred lines that differ from the superior single-cross parent at 1-3 loci to create a composite parent. While retaining the uniformity, stability, and specificity of the original single-cross, the improved composite parent plants are robust and have higher yields, thereby increasing the seed production of the superior single-cross. If the loci difference is large, the improved single-cross will have poor uniformity, stability, and specificity, and cannot be used as an improved variety of the superior single-cross.

[0042] 4. Analysis of combining ability of closely related self-crossed lines

[0043] In 2019, winter sowing was carried out at the Sanya Nanfan Base. 50-100 plants of HB12, HB37, HB52, JunM6968 and JunDan58 paternal parent Jun262 were sown respectively. Three closely related inbred lines HB12, HB37, HB52 and the superior single-cross hybrid JunM6968 (A1) were used as female parents (♀), and the superior single-cross hybrid Jun262 (B) was used as male parent (♂) as a test species to create single-cross hybrids. In the summer of 2020, 50-100 plants of four single-cross hybrids were sown. Combining ability analysis of the hybrids revealed that HB37(♀) (pedigree: PH6WC / PH09B / / Zheng58) showed the best combining ability with Jun262(♂). The combining ability of HB37(♀) × Jun262(♂) was comparable to that of JunM6968(♀) × Jun262(♂). Finally, the inbred line HB37(A2), a close relative of JunM6968, was deemed suitable for improving seed production yield of single-cross hybrids.

[0044] Among them, Jun 262 and Jun M6968 are not the same type of plant; they are the male and female parents of the single-cross hybrid Jun Dan 58, respectively. This application uses Jun 262, the male parent of Jun Dan 58, as a test species to test the combining ability of the female parent Jun M6968, and screens out female parents with the same combining ability as the male parent of Jun Dan 58. Only by using female parents with the same combining ability to form a composite female parent can we ensure that the composite parent also has a high combining ability with the original male parent Jun 262, without improving the basic traits of Jun Dan 58. Furthermore, a female parent with the same combining ability as the same male parent is a prerequisite for improving the seed production yield of single-cross hybrids from closely related inbred lines. After forming a composite female parent, the hybrid formed with the male parent Jun 262 is basically consistent with the original single-cross hybrid Jun Dan 58 in terms of yield and other traits. At the same time, screening out inbred lines with the same combining ability as the original superior inbred lines from multiple closely related inbred lines can greatly reduce the workload of subsequent work.

[0045] Example 2: Composite parental pairing and improved superior single crosses

[0046] 1. Combination of composite parent plants

[0047] In the summer of 2021, 200-300 homozygous parent plants, namely Jun M6968 (A1) and closely related inbred line HB37 (A2), were sown. During the pollen shedding and silking stage, 100 plants of A1 and A2 were selected for self-pollination to obtain homozygous female parents A1 and A2. Following reciprocal crosses, pollen from 10-30 robust, sexually coordinated, and high-pollen-producing superior plants (as male parents) were selected from A1, mixed evenly, and applied to A1 plants. 2. The remaining 100-200 individual plants (as female parent) are pollinated and cross-pollinated according to the orthogonal cross-pollution method A2(♀)×A1(♂) to obtain composite female parent A3; 10-30 superior individual plants (as male parent (♂)) from A2 are selected, their pollen is mixed evenly, and the remaining 100-200 individual plants (as female parent (♀)) of A1 are pollinated and cross-pollinated according to the reciprocal cross-pollution method A1(♀)×A2(♂) to obtain composite female parent A4.

[0048] 2. Improved superior single-cross hybrid Jundan 58

[0049] In the winter of 2021, at the Nanfan Breeding Base in Sanya City, homozygous female parents (A1, A2) and composite female parents (A3, A4) were used as female parents, and the superior single-cross cultivar Jun 262(B) was used as the male parent. Single-cross cultivars were then created: A1(♀)×B(♂), A2(♀)×B(♂), A3(♀)×B(♂), and A4(♀)×B(♂). Sowing was carried out with a female parent:male parent row ratio of 3:1. Each of the four female parents was planted in 20 rows, with a row length of 10m, a row spacing of 0.6m, and a planting density of 7.5×10⁶. 4 Plant / hm 2Before the female parent tassels emerged, the male ears of four female parents were removed, and open-field pollination was performed to obtain the original single-cross hybrid Jundan 58(H1)(A1(♀)×B(♂)) and improved single-cross hybrids H2(A2(♀)×B(♂)), H3(A3(♀)×B(♂)), and H4(A4(♀)×B(♂)). The removal of male ears from all female parents was done to ensure the purity of the offspring.

[0050] Among them, Jundan 58 (H1) is a newly approved maize variety. This application mainly focuses on the seed production yield of this variety. The advantage of this application is that it can increase the seed production yield of Jundan 58 without changing its traits.

[0051] Example 3 Evaluation of maternal inbred lines

[0052] 1. Identification of SSR molecular markers

[0053] In the summer of 2022, 200-300 plants of each of the four maternal parents (A1, A2, A3, and A4) were sown. When the maize reached the three-leaf stage, 10 leaves from each material were collected and mixed. Molecular identification was performed using 20 pairs of core maize SSR primers published in GB / T 39914-2021. Specificity analysis was conducted on the four maternal inbred lines. The operation procedure for the SSR molecular marker technology was the same as in Example 1. The results are shown in Table 1. Homozygous parents A1 and A2 showed two loci differences in primers umc1432 and umc1125, while A3 and A4 showed one locus difference in primer phi080. A1 and A2 differed from A3 and A4 in three loci from primers umc1432, umc1125, and phi080.

[0054] Table 1. Molecular differences among the four inbred lines and four hybrids.

[0055]

[0056] 2. Field trait testing

[0057] In the summer of 2022, 200-300 plants of each of the four parent lines were sown. After emergence of seedlings in varieties A1, A2, A3, and A4, 40 plants were randomly selected from each plot and marked. Using the Guidelines for Testing Variety Specificity, Uniformity and Stability (DUS) of Maize published in GB / 19557.24-2018, 39 mandatory traits were selected from 60 maize varieties for field uniformity and specificity analysis. The four maternal inbred lines were also tested. The results are as follows: Figs. 1-3As shown, there is no specificity between the two homozygous inbred lines A1 and A2, and also no specificity between the two compound inbred lines A3 and A4. However, the compound inbred lines A3 and A4 exhibit specificity with the homozygous inbred lines A1 and A2 in three traits (plant height at maturity, ear height to plant height ratio, and number of grains per ear) in both plant and ear. Among these, ♀-1, ♀-2, ♀-3, and ♀-4 are A1, A2, A3, and A4, respectively.

[0058] from Figs. 1-3 It can be seen that the seedling traits of the four inbred lines A1, A2, A3, and A4 are consistent: the tip of the first leaf is round in all four lines, the anthocyanin color intensity of the first leaf sheath is strong in all four lines, and the greenness of the seedling leaves is medium in all four lines. During the silking and pollination stage, the leaf length and width of the four inbred lines are consistent, but the plant height of A1, A2, and A3 is lower than that of A4. The ear traits of the four inbred lines A1, A2, A3, and A4 are similar: the kernel color is uniformly medium yellow, the kernel shape is relatively hard, the kernel shape is nearly round, and the anthocyanin color intensity of the glumes is either negligible or very weak; however, the composite parent inbred lines A3 and A4 have more kernels per row than the homozygous parent inbred lines A1 and A2.

[0059] Four improved varieties were compared with the original varieties.

[0060] 1. Identification of SSR molecular markers

[0061] In the summer of 2022, 200-300 plants of each of the four single-cross varieties (H1, H2, H3, and H4) were sown. When the maize reached the three-leaf stage, leaves from 10 plants of each material were collected and mixed. Using the 20 pairs of core SSR primers for maize published in GB / T 39914-2021, specificity analysis was performed between the improved varieties H2, H3, and H4 and the original variety Jundan 58 (H1). The procedure for using the SSR molecular markers was the same as in Example 1. The results are shown in Table 2. There were 2-3 differentially expressed sites between the improved varieties H2, H3, and H4 and the original variety Jundan 58 (H1).

[0062] Table 2. Molecular differences among the four inbred lines and four hybrids.

[0063]

[0064] 2. Field trait testing

[0065] In the summer of 2022, 200-300 plants of each of the four single-cross varieties were sown. After emergence of H1, H2, H3, and H4 seedlings, 40 plants were randomly selected from each plot and marked. Using the Guidelines for Testing Variety Specificity, Uniformity and Stability (DUS) of Maize published in GB / 19557.24-2018, 39 mandatory traits were selected from 60 maize varieties for field uniformity and specificity analysis. The improved varieties were compared with the original varieties. The results showed that the 39 phenotypic traits of the three improved single-cross varieties H2, H3, and H4 were identical to those of the original single-cross variety Jundan 58 (H1), with no specificity.

[0066] 3. Comparison of seed production yield and its components

[0067] In the summer of 2022, four hybrid varieties of maize were harvested at maturity. Ten sample ears were selected from each plot based on the average ear weight (weight of 10 sample ears = (total harvested ear weight / total harvested ears) × 10) to investigate yield composition and ear traits, primarily focusing on yield and kernel count comparisons. All harvested ears from each plot were threshed, naturally air-dried, and then the kernel moisture content was measured. Yield per unit area was calculated based on a 14% moisture content. The results are shown in Table 3. The yield difference among the four hybrid varieties was less than 5.0%, indicating that the yield difference was caused by production errors rather than variations in varietal yield characteristics.

[0068] Table 3 Comparison of Yield and Yield Components of Four Hybrids

[0069]

[0070] Example 5: Comparison of the yield and yield components of four female parent lines

[0071] In March 2022, at the Sanya Nanfan Breeding Base, after varieties H1, H2, H3, and H4 matured, ten sample ears were selected from the harvest area to examine yield composition and ear traits. These samples were compared with the original single crosses to select superior germplasm. The remaining ears were threshed and naturally air-dried. After drying, the grain moisture content was measured, and the yield per unit area was calculated based on a moisture content of 14%.

[0072] The results are shown in Table 4. The seed production of the two composite female parents, A3 and A4, was significantly higher than that of the homozygous parents, A1 and A2. The average seed production of A3 was 23.2% higher than that of JunM6968(A1) ((7033.4-5708.6) / 5708.6×100%), and the number of grains per ear was 32.5% higher ((15.4×28.5-16.0×20.7) / (16.0×20.7)×100%).

[0073] Table 4 Comparison of seed production yield and yield components of the four inbred lines

[0074]

[0075] The above results indicate that the use of closely related inbred lines from JunM6968 to improve the seed production yield of the single-cross hybrid JunDan58 is effective. This application found that although there were 1-3 molecular differences among the four single-cross hybrids, there were no morphological differences, indicating good uniformity, and they can be considered the same variety. There are two key aspects in this application: first, the screening and identification of closely related inbred lines; and second, the identification of the uniformity and specificity of the improved single-cross hybrids. Only closely related inbred lines with a gene locus difference of <3 can produce improved single-cross hybrids with uniformity, lacking specificity and innovation, and resembling the original single-cross hybrids. Only such closely related inbred lines can be used to improve the seed production yield of single-cross hybrids.

[0076] In summary, improving the maternal parent using closely related inbred lines of superior single-cross maternal parents can enrich the genetic information of the maternal parent and create superior germplasm. However, if the loci differences between closely related inbred lines are large, the uniformity and stability of the offspring single-cross maternal parents are poor. This application selects the superior single-cross maternal parent Jundan 58 (National Approval No. 20210073) as the target, and uses closely related inbred lines with 1-3 loci differences from its maternal parent JunM6968 to create a composite maternal parent. While preserving the uniformity, stability, and specificity of Jundan 58, the use of closely related inbred lines not only enriches the genetic information of the maternal parent, but also results in robust composite maternal parent plants with higher yields, thereby increasing maternal seed production. The method for increasing maternal seed production in this application is highly practical, operable, targeted, cost-effective, and significantly improves seed production efficiency, and can be widely applied in maternal seed production.

Claims

1. A method for improving the seed yield of maize single-cross hybrids using closely related inbred lines, characterized in that: Using superior single-cross hybrid female parents as the research object, through germplasm resource collection and screening, closely related inbred lines with 1-3 molecular differences from the superior single-cross hybrid female parents were screened out, and composite female parents were bred using these closely related inbred lines; through the improvement of superior single-cross hybrids, varieties consistent with the composite female parents and single-cross hybrids were selected; and then their yield and yield composition traits were evaluated. The method for improving the yield of maize single-cross seed production using closely related inbred lines includes the following steps: (1) Selection of superior single-cross varieties: Select single-cross varieties with excellent traits such as high yield, wide adaptability, stress resistance, high quality and high resistance; (2) Collection and identification of closely related inbred lines: Based on the superior single-cross maternal parent A1, the maternal parent was classified into groups using SSR molecular marker technology; germplasm resources of the same group as the superior single-cross maternal parent A1 were collected, and the specificity analysis between the collected germplasm resources and A1 was performed using SSR molecular marker technology to screen out closely related inbred lines that have 1-3 molecular differences from the superior single-cross maternal parent A1; (3) Combining ability determination and analysis of closely related inbred lines: Using the superior single cross male parent B as the test species, and the collected closely related inbred lines and the superior single cross female parent A1 as the female parent, single crosses were bred. The combining ability of the bred single crosses was determined and analyzed, and closely related inbred lines A2 with the same high combining ability as A1♀×B♂ were screened. (4) Combination of composite female parents: Sow seeds of A1 and A2, and obtain composite female parents A3 and A4 by orthogonal combination A2♀×A1♂ and reciprocal combination A1♀×A2♂. (5) Improvement of superior single crosses: Sow homozygous female parents A1 and A2, composite female parents A3 and A4 and superior single cross male parent B. Use A1, A2, A3 and A4 as female parents and superior single cross B as male parent to fertilize single crosses A1♀×B♂, A2♀×B♂, A3♀×B♂ and A4♀×B♂ to obtain single cross H1 and improved single crosses H2, H3 and H4 respectively. (6) Evaluation of parents and superior single crosses: Four female parents A1, A2, A3, A4 and four single crosses H1, H2, H3, H4 were sown respectively. The consistency and specificity of the four female parents and four single crosses were analyzed using maize SSR molecular marker technology and DUS test technology. (7) Seed production yield comparison: The yield and yield composition traits of the single crosses H1, H2, H3 and H4 obtained in step (5) were evaluated. The superior single-cross female parent A1 is the inbred line JunM6968 of JunDan58, and A2 is the closely related inbred line HB37 of JunM6968. The pedigree is: PH6WC / PH09B / / Zheng58. The superior single-cross male parent B is the male parent Jun262 of JunDan58. H1 is the improved superior single-cross male parent JunDan58.

2. The method for improving the yield of maize single-cross seed production using closely related inbred lines as described in claim 1, characterized in that, The method for assembling the composite female parent in step (4) is as follows: Sow 200-300 homozygous parent plants A1 and A2 respectively. During the pollen shedding and silking period, select 100 plants of A1 and A2 respectively for self-pollination to obtain homozygous female parents A1 and A2. Select 10-30 superior single plants with strong plants, coordinated male and female reproductive functions, and large pollen production from A1, mix them evenly, and pollinate the remaining 100-200 single plants of A2 to obtain composite female parent A3. Select 10-30 superior single plants with pollen from A2, mix them evenly, and pollinate the remaining 100-200 single plants of A1 to obtain composite female parent A4.

3. The method for improving the yield of maize single-cross seed production using closely related inbred lines as described in claim 1, characterized in that, The specific method for improving the superior single cross in step (5) is as follows: using homozygous maternal parents A1, A2, and composite maternal parents A3 and A4 as maternal parents, and superior single cross B as paternal parents, sowing is carried out with a row ratio of maternal parent: paternal parent = 3:

1. The male ears of the maternal parents are removed before the female parents tassel, and open isolation pollination is carried out to obtain the original single cross H1 and improved single cross H2, H3, and H4.

4. The method for improving the yield of maize single-cross seed production using closely related inbred lines as described in claim 1, characterized in that, The evaluation method for the parents and superior single crosses in step (6) is as follows: 200-300 plants of the four maternal parents A1, A2, A3, A4 and the four single crosses H1, H2, H3, H4 are sown respectively. When the maize grows to the 3-leaf stage, 10 leaves from each material are taken and mixed. Molecular identification is performed using the 20 pairs of core SSR primers published in GB / T39914-2021. After the emergence of A1, A2, A3, A4 and H1, H2, H3, H4, 40 plants are randomly selected from each area and marked. According to the plant variety specificity, uniformity and stability test guidelines published in GB / 19557.24-2018, 39 mandatory traits are selected from 60 maize varieties in the DUS test items to analyze and identify the uniformity and specificity of the four maternal parents and the four single crosses.

5. The method for improving the yield of maize single-cross seed production using closely related inbred lines as described in claim 1, characterized in that, The specific method for comparing seed production yield in step (7) is as follows: during the corn seed maturity period in step (5), harvest 4 hybrid seeds, select 10 sample ears from each area, examine the yield composition and ear traits, compare and analyze with the original single cross, and select superior germplasm.

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