Method for creating N-type cytoplasm feed beet three-line germplasm resources by using N-type cytoplasm sugar beet maintainer line

Abstract

The invention provides a method for creating an N-type cytoplasm feed beet three-line germplasm resource by using a beet maintainer line for N-type cytoplasm sugar. By means of the method, a large number of N-type cytoplasm sugar beet germplasm resources can be made into N-type feed beet germplasm resources, increase of the N-type feed beet germplasm resources is achieved, creation of feed beet three-line germplasm resources is accelerated, and feed beet breeding is better carried out.

Description

Technical Field

[0001] The present application relates to the technical field of crop breeding, and in particular to a method for creating three-line germplasm resources of N-type cytoplasmic fodder beet by utilizing a maintainer line of N-type cytoplasmic sugar beet. Background Art

[0002] At present, beet breeding work at home and abroad is mostly concentrated on sugar beets, while the investment in breeding work for fodder beets is relatively low and the germplasm resources are relatively scarce. Most of the superior hybrids of beets are three-way hybrids, namely sterile lines, maintenance lines, and pollination lines. With the advancement of existing biological breeding technology, after identifying the cytoplasm and nuclear genotypes of existing fodder beets, it was found that there are relatively few N-type cytoplasm germplasm resources. The existing breeding of fodder beets is still conventional single-cross breeding. Beet breeding is carried out through three-way hybrid breeding, which can better improve the growth, yield and quality of the variety. However, the most critical link in the three-way hybrid is the breeding of sterile lines and maintenance lines. Due to the scarcity of N-type cytoplasm germplasm resources of fodder beets, the breeding difficulty of maintenance lines and N-type pollination lines is increased. At present, the methods for innovation of fodder beet germplasm resources mostly utilize conventional breeding and creation with existing fodder beet germplasm resources. Due to the scarcity of existing N-type cytoplasm germplasm resources of fodder beet, the number of creations is small and the germplasm is prone to degeneration, which seriously restricts the use of fodder beet. Summary of the invention

[0003] In view of the above-mentioned technical limitations, the present application proposes a method for creating a three-line germplasm resource of N-type cytoplasmic fodder beet using an N-type cytoplasmic sugar beet maintainer line; it overcomes the deficiencies and defects mentioned in the background technology.

[0004] To achieve the above objectives, this application adopts the following technical solutions: The invention of the present application is to provide a method for creating N-type cytoplasmic feed beet three-line germplasm resources by using N-type cytoplasmic sugar beet maintainer line, comprising the following steps: S1. Select multiple N-type cytoplasmic sugar beet maintenance lines for mixed sowing to establish a mixed population, and harvest the population mother root B; hybridize the population mother root B with multiple selected fodder beet pollination lines C to obtain F1 generation hybrids; S2. Sow F1 hybrid seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, observe the root bark color with the naked eye, eliminate the white ones, and keep the mother roots with color; backcross with fodder beet pollination line C the next year. Backcrossing means planting the mother roots with color harvested from the F1 hybrid seeds and the fodder beet pollination line C in a spatial isolation area, open pollination, mixed sowing, and harvesting the backcross first generation mother parent seeds BC1F1; S3. Sow BC1F1 seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, observe the root bark color with the naked eye, eliminate the white ones, and keep the mother roots with purer colors; the next year, backcross with the fodder beet pollination line C. Backcrossing is to BC1F1 The colored mother roots harvested from the first generation hybrid seeds were planted in a spatial isolation area with the fodder beet pollination line C, open pollinated, mixed sown, and the first generation backcross female parent seeds BC2F1 were harvested; S4. Sow BC2F1 seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, observe the root bark color with the naked eye, eliminate the white ones, and retain the mother roots with purer colors to obtain N-type cytoplasm forage beet germplasm resources; plant the mother roots in the second year, and harvest the N-type cytoplasm germplasm resources from the mother roots. If the color is impure, repeat steps S2 and S3; S5. The N-type cytoplasmic feed beet germplasm resources were identified by molecular marker technology, and the N-type cytoplasmic feed beet containing pure recessive genotype xxzz was reused as the proposed maintainer line B; the N-type cytoplasmic feed beet with a dominant gene X was reused as the proposed pollination line C; the molecular marker technology used primer TR1 acting on the VNTR site of beet mitochondria to identify the cytoplasmic type of beet, and the polymorphism of the upstream region of bvORF17 of nuclear DNA was used to identify the nuclear genotype (method reference: 1. Taguchi K, Hiyama H, Yui-Kurino R, et al. HybridBreeding Skewed the Allelic Frequencies of Molecular Variants Derived fromthe Restorerof fertility 1 Locus for Cytoplasmic Male Sterility in Sugar Beet(Beta vulgaris L.) [J]. Crop Science, 2014, 54(4): 1407-1412. 2.Nishizawa S, Kubo T, Mikami T. Variable number oftandem repeat loci in the mitochondrialgenomes of beets [J]. Current genetics, 2000, 37(1): 34-38.).

[0005] Breeding of sterile lines and maintainer lines: Plant the sterile line ms of forage beet with the identified maintainer line B in a centralized isolation area for plant-to-plant hybridization, harvest seeds from sterile plants and maintainers respectively, harvest hybrid seeds msF1 from sterile plants, and harvest self-fertilized seeds B-1 from maintainers; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, yield and quality traits, and select paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, brown spot, high sugar content, small leaf clusters, smooth roots, small root heads, and strong stress resistance for reuse; (Method reference book: Cui Ping, "Beet Germplasm Resource Description Specifications and Data Standards", Beijing: China Agriculture Press, 2006.) Pollination line selection: self-pollinate the identified pollination line C to harvest the self-pollination first generation C-1 seeds; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, yield and quality traits, and select paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, brown spot, high sugar content, small leaf clusters, smooth roots, small root heads, and strong stress resistance for reuse; Breeding of sterile lines and maintainer lines: backcross the harvested msF1 with B-1 and plant them in a centralized isolation area. Investigate fertility when flowering and observe anther morphology. Plants with plump yellow pollen are fertile plants, while plants with small, shriveled, light yellow or transparent pollen without vitality are sterile plants. Select sterile plants of msF1 and perform plant-to-plant hybridization with B-1. Harvest seeds from sterile plants and maintainer plants respectively. The first-generation backcross seeds harvested from sterile plants are msBC1F1, and the self-fertilized seeds B-1-1 are harvested from the maintainer plants. Sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, and brown spot, high sugar content, small leaf clusters and leaves, smooth root shape, small root heads, and strong stress resistance for reuse. S7. Pollination line breeding: The harvested C-1 mother roots are self-pollinated individually to harvest the second-generation C-1-1 seeds; sowed in the summer of the same year, and the disease resistance, stress resistance, field biological traits, agronomic traits, yield and quality traits are identified, and paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, brown spot, high sugar content, small leaf clusters, smooth roots, small root heads, and strong stress resistance are selected for reuse; Breeding of sterile lines and maintainer lines: backcross the harvested msBC1F1 with B-1-1, plant them in a centralized isolation area for plant-to-plant hybridization, harvest seeds from sterile plants and maintainer plants respectively, the backcross second-generation seeds harvested from sterile plants are msBC2F1, and the self-fertilized seeds B-1-1-1 are harvested from the maintainer plants; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, yield and quality traits, and select paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, brown spot, high sugar content, small leaf clusters and leaves, smooth root shape, small root heads, and strong stress resistance for reuse; S8. Pollination line breeding: The harvested C-1-1 mother roots are self-pollinated individually to harvest the third generation of self-pollinated C-1-1-1 seeds; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, yield and quality traits, and select paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, brown spot, high sugar content, small leaf clusters, smooth roots, small root heads, and strong stress resistance for reuse; S9. Breeding of sterile lines and maintainer lines: backcross the harvested msBC2F1 with B-1-1-1, plant them in a centralized isolation area for plant-to-plant hybridization, harvest seeds from the sterile plants and maintainer plants respectively, the backcross third-generation seeds harvested from the sterile plants are msBC3F1, and the self-fertilized seeds B-1-1-1-1 are harvested from the maintainer plants; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits such as resistance to root rot, rhizomes, powdery mildew, and brown spot, high sugar content, small leaf clusters and leaves, smooth root shape, small root heads, and strong stress resistance for reuse; Pollination line selection: By self-pollinating the harvested C-1-1-1 mother roots, you can harvest pollination lines with consistent comprehensive traits.

[0006] Breeding of sterile lines and maintainer lines: backcross the harvested msBC3F1 with B-1-1-1-1, and harvest seeds from the sterile plants and maintainer plants respectively. The fourth-generation backcross seeds harvested from the sterile plants are the sterile line, and the self-pollinated seeds harvested from the maintainer plants are the homogeneous maintainer line of the sterile line.

[0007] Compared with the prior art, this application has the following advantages: 1. The present invention firstly uses different types of sugar beet maintainer lines as mother parents. The sugar beet maintainer lines have more numbers, which will provide more possibilities for the creation of N-type cytoplasm in fodder beets.

[0008] 2. The present invention firstly uses multiple different types of sugar maintenance lines to establish a mixed population, harvests the mother roots B of the population, and selects multiple fodder beet pollination lines C with excellent comprehensive traits for hybridization. In this way, different types of hybrid combinations can be configured, and hybrid materials with different phenotypes can be harvested, so as to better screen germplasm resources that meet the breeding target phenotype.

[0009] 3. The present invention adopts the backcrossing method when replacing the sugar beet maintainer line, which can better transform the unique phenotype of sugar beet and obtain the performance characteristics closer to fodder beet.

[0010] 4. The present invention uses a molecular marker method to accurately identify the cytoplasm and nuclear genotypes of the newly obtained fodder beet N-type cytoplasm germplasm resources, which can more quickly select and breed pure line materials.

[0011] 5. The present invention obtains pure line materials with relatively stable genetics and phenotypes, namely, maintainer lines and pollination lines, through multi-generation self-pollination while obtaining the intended maintainer lines and pollination lines. By backcrossing the intended maintainer lines with the fodder sterile lines for four generations, multiple pairs of sterile lines and maintainer lines can be obtained. At the same time, the sterile lines and maintainer lines can also be used to configure multiple different types of binary sterile lines, which enriches the existing germplasm resource bank and provides more possibilities for the breeding of fodder beets. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown is a flow chart of a method for creating three-line germplasm resources of N-type cytoplasmic fodder beet using N-type cytoplasmic sugar beet maintainer lines in one embodiment of the present application. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0014] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0015] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0016] Example 1 1. The first year: Select the sugar maintenance line B with excellent traits and the mixed fodder beet pollination line C with various phenotypes to cultivate mother roots in late July or early August, and harvest the mother root materials in early November.

[0017] 2. The second year: Plant the mother root materials separately in spring, use the mixed group sugar maintenance line B as the female parent; and plant it with the fodder beet pollination line C as the male parent in a spatial isolation area for hybridization. Harvest the mixed F1 hybrids from the female parent. Sow F1 seeds in late July and early August of the same year. When harvesting the mother roots, investigate the plant phenotype, eliminate the white ones, and retain the mother roots that show color.

[0018] 3. The third year: In spring, the harvested F1 generation female roots are planted in a spatial isolation area with the fodder beet pollination line C for backcrossing. The seeds of the first generation of backcrossing (BC1F1) are harvested. In summer, BC1F1 seeds are sown. When the mother roots are harvested, the phenotype of the plants is investigated, white ones are eliminated, and the mother roots with purer colors are retained.

[0019] 4. The fourth year: plant the mother roots of BC1F1 and the fodder beet pollination line C in a spatial isolation area for backcrossing. Harvest the seeds of the second generation of backcrossing (BC2F1). Sow BC2F1 seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, eliminate the white and impure mother roots, and repeat the steps of the second and third years. Keep the pure red mother roots, and then you will get a large number of fodder beet N-type cytoplasm germplasm resources. Use molecular marker technology to identify the retained N-type fodder beet cytoplasm germplasm resources, and reuse the identified N (xxzz) as the proposed maintenance line B. N (X) is reused as the proposed pollination line C.

[0020] 5. The fifth year: Breeding of sterile lines and maintainer lines: The sterile line ms of fodder beet was hybridized with the identified maintainer line B in a plant-to-plant hybridization, and seeds were harvested from the sterile plants and maintainer plants respectively. The hybrid harvested from the sterile plants was msF1, and the self-fertilized seeds B-1 were harvested from the maintainer plants. Sow in the summer of the same year, identify their phenotypes and disease resistance, and select paired materials with excellent comprehensive traits for reuse.

[0021] Pollination line selection: The identified pollination line C is self-pollinated individually to harvest the self-pollinated first generation C-1 seeds. Sow in the summer of the same year, identify its phenotype and disease resistance, and select materials with excellent comprehensive traits for reuse.

[0022] 6. The sixth year: Breeding of sterile lines and maintainer lines: Harvest msF1 and B-1 for plant-to-plant backcrossing, harvest seeds from sterile plants and maintainer plants respectively, the first generation of backcross seeds harvested from sterile plants are msBC1F1, and the self-fertilized seeds B-1-1 are harvested from maintainer plants. Sow in the summer of the same year, identify their phenotypes and disease resistance, and select paired materials with excellent comprehensive traits for reuse.

[0023] Pollination line selection: The harvested C-1 mother roots are self-pollinated individually to harvest the second generation C-1-1 seeds. They are sown in the summer of the same year, their phenotypes and disease resistance are identified, and materials with excellent comprehensive traits are selected for reuse.

[0024] 7. Seventh year: Breeding of sterile lines and maintainers: Harvest msBC1F1 and B-1-1 for plant-to-plant backcrossing, harvest seeds from sterile plants and maintainers respectively, the second generation of backcross seeds harvested from sterile plants are msBC2F1, and the self-fertilized seeds B-1-1-1 are harvested from maintainers. Sow in the summer of the same year, identify their phenotypes and disease resistance, and select paired materials with excellent comprehensive traits for reuse.

[0025] Pollination line selection: The harvested C-1-1 mother roots are self-pollinated individually, and the self-pollinated third-generation C-1-1-1 seeds are harvested. Sow in the summer of the same year, identify their phenotypes and disease resistance, and select materials with excellent comprehensive traits for reuse.

[0026] 8. The eighth year: Breeding of sterile lines and maintainer lines: Harvest msBC2F1 and B-1-1-1 for plant-to-plant backcrossing, harvest seeds from sterile plants and maintainer plants respectively, the third generation of backcross seeds harvested from sterile plants are msBC3F1, and the self-fertilized seeds B-1-1-1-1 are harvested from maintainer plants. Sow in the summer of the same year, identify their phenotypes and disease resistance, and select paired materials with excellent comprehensive traits for reuse.

[0027] Pollination line selection: The harvested C-1-1-1 mother roots are self-pollinated individually to obtain pollination lines with consistent comprehensive traits.

[0028] 9. The ninth year: Breeding of sterile lines and maintainer lines: Harvest msBC3F1 and B-1-1-1-1 for plant-to-plant backcrossing, and harvest seeds from the sterile plants and maintainer plants respectively. The fourth-generation backcross seeds harvested from the sterile plants have excellent comprehensive traits and can stably inherit the sterile line. The self-pollinated seeds harvested from the maintainer plants are the homogeneous maintainer line of the sterile line.

[0029] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for creating three-line germplasm resources of N-type cytoplasmic fodder beet using N-type cytoplasmic sugar beet maintainer lines, characterized in that: The following steps are involved: S1. Select multiple N-type cytoplasmic sugar beet maintenance lines for mixed sowing to establish a mixed population, and harvest the population mother root B; hybridize the population mother root B with multiple selected fodder beet pollination lines C to obtain F1 generation hybrids; S2. Sow F1 hybrid seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, observe the root bark color with the naked eye, eliminate the white ones, and keep the mother roots with color; backcross with fodder beet pollination line C the next year. Backcrossing means planting the mother roots with color harvested from the F1 hybrid seeds and the fodder beet pollination line C in a spatial isolation area, open pollination, mixed sowing, and harvesting the backcross first generation mother parent seeds BC1F1; S3. Sow BC1F1 seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, observe the root bark color with the naked eye, eliminate the white ones, and retain the mother roots with purer colors; backcross with fodder beet pollination line C the next year. Backcrossing is to plant the mother roots with color harvested from the BC1F1 hybrid seeds and the fodder beet pollination line C in a spatial isolation area, open pollinate, mix sow, and harvest the backcross first generation mother parent seeds BC2F1; S4. Sow BC2F1 seeds in summer, investigate the phenotype of the plants when harvesting the mother roots, observe the root bark color with the naked eye, eliminate the white ones, and retain the mother roots with purer colors to obtain N-type cytoplasm forage beet germplasm resources; plant the mother roots in the second year, and harvest the N-type cytoplasm germplasm resources from the mother roots. If the color is impure, repeat steps S2 and S3; S5. Using molecular marker technology to identify the N-type cytoplasmic feed beet germplasm resources, the identified N-type cytoplasmic feed beet containing pure recessive genotype xxzz is reused as the intended maintainer line B; the N-type cytoplasmic feed beet with at least one dominant gene X is reused as the intended pollination line C; S6. Breeding of sterile lines and maintainer lines: Plant the sterile line ms of forage beet and the identified maintainer line B in a centralized isolation area for plant-to-plant hybridization, harvest seeds from the sterile plants and maintainer plants respectively, harvest hybrid seeds msF1 from the sterile plants, and harvest self-fertilized seeds B-1 from the maintainer plants; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits for reuse; Pollination line breeding: self-pollinate the identified pollination line C and harvest the self-pollinated first-generation C-1 seeds; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, yield and quality traits, and select paired materials with excellent comprehensive traits for reuse; S7. Breeding of sterile lines and maintainer lines: backcross the harvested msF1 with B-1 and plant them in a centralized isolation area. Investigate fertility when flowering and observe anther morphology. Plants with plump yellow pollen are fertile plants, while plants with small, shriveled, light yellow or transparent pollen without vitality are sterile plants. Select sterile plants of msF1 and perform plant-to-plant hybridization with B-1. Harvest seeds from sterile plants and maintainer plants respectively. The backcross first-generation seeds harvested from sterile plants are msBC1F1, and the self-fertilized seeds B-1-1 are harvested from maintainer plants. Sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits for reuse. Pollination line breeding: The harvested C-1 mother roots are self-pollinated individually to harvest the second-generation C-1-1 seeds; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits for reuse; S8. Breeding of sterile lines and maintainer lines: backcross the harvested msBC1F1 with B-1-1, plant them in a centralized isolation area for plant-to-plant hybridization, harvest seeds from sterile plants and maintainer plants respectively, the backcross second-generation seeds harvested from sterile plants are msBC2F1, and the self-fertilized seeds B-1-1-1 are harvested from the maintainer plants; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits for reuse; Pollination line breeding: The harvested C-1-1 mother roots are self-pollinated individually to harvest the third generation of self-pollinated C-1-1-1 seeds; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits for reuse; S9. Breeding of sterile lines and maintainer lines: backcross the harvested msBC2F1 with B-1-1-1, plant them in a centralized isolation area for plant-to-plant hybridization, harvest seeds from sterile plants and maintainer plants respectively, the third-generation backcross seeds harvested from sterile plants are msBC3F1, and the self-fertilized seeds B-1-1-1-1 are harvested from the maintainer plants; sow in the summer of the same year, identify disease resistance, stress resistance, field biological traits, agronomic traits, and yield and quality traits, and select paired materials with excellent comprehensive traits for reuse; Pollination line selection: self-pollinate the harvested C-1-1-1 mother roots to obtain pollination lines with consistent comprehensive traits; S10. Breeding of sterile lines and maintainer lines: backcross the harvested msBC3F1 with B-1-1-1-1, and harvest seeds from the sterile plants and maintainer plants respectively. The fourth-generation backcross seeds harvested from the sterile plants are the sterile lines, and the self-pollinated seeds harvested from the maintainer plants are the homogeneous maintainer lines of the sterile lines.

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