A method for creating monoembryonic sterile lines and maintainer lines of red sugar beet

Abstract

The present invention provides a method for creating a single-embryonic sterile line and maintainer line of red beet, comprising: sowing imported monoembryonic red beet varieties and domestic polyembryonic red beet lines, identifying them using molecular marker technology and morphology, and introducing a single-embryonic gene and a nuclear male sterile gene into the red beet lines through hybridization and selfing to create a single-embryonic maintainer line of red beet; simultaneously, selfing the monoembryonic red beet varieties to produce a single-embryonic sterile line of red beet; and continuously pollinating the sterile line while selfing the maintainer line, ultimately releasing pairs of single-embryonic sterile lines and maintainer lines of red beet. The present invention can increase the number of single-embryonic cytoplasmic male sterile lines and maintainer lines of red beet in my country from zero to a large number, and achieve a single-embryonic rate of more than 98% for the single-embryonic red beet maintainer line seeds, a single-embryonic rate of more than 98% for the sterile line seeds, and a sterility rate of 100%. This provides support for the future large-scale creation of single-embryonic cytoplasmic male sterile lines and maintainer lines of monoembryonic red beet, as well as the creation of new single-embryonic hybrid red beet varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant strain improvement, and more particularly relates to a method for creating a monoembryonic sterile line and a maintainer line of red sugar beet. Background Art

[0002] Edible red beet (Beta vulgaris subsp. vulgaris L.) belongs to the Amaranthaceae family and the Beta genus. It is one of four varieties of the common beet subspecies. Edible red beet is entirely red and is also known as purple beet, red beet, vegetable beet, or flame beet. Over the course of long evolutionary processes, the Beta genus has gradually differentiated into diverse species and diverse eco-geographical types and morphological characteristics, influenced by geographical isolation or cross-pollination. Due to its rich nutritional value and its potential to prevent cancer and lower blood lipids, blood sugar, and blood pressure, the cultivated area in my country has been increasing annually.

[0003] At present, all monoembryonic red beet varieties used in production in my country are imported from abroad. China only has polyembryonic red beet germplasm resources, but no monoembryonic red beet resources. To cultivate monoembryonic red beet varieties, it is necessary to use monoembryonic red beet cytoplasmic male sterile lines and maintainer lines. The present invention discloses a method for creating a monoembryonic red beet cytoplasmic male sterile line and maintainer line. The method uses monoembryonic red beet varieties imported from abroad, combines molecular marker technology and morphological identification, and introduces monoembryonic genes and nuclear male sterile genes into domestic red beet lines through hybridization and selfing to create a red beet monoembryonic maintainer line. At the same time, the foreign monoembryonic red beet varieties are selfed to produce a red beet monoembryonic sterile line. The maintainer line is selfed while continuously pollinating the sterile line, and finally, pairs of red beet monoembryonic sterile lines and maintainer lines are released. This method can create multiple pairs of red beet monoembryonic sterile lines and maintainer lines. The present invention can increase the number of monoembryonic sterile lines and maintainer lines of red beets in my country from scratch, and provide technical support for breeding monoembryonic red beet varieties with independent intellectual property rights in my country. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for creating a monoembryonic sterile line and a maintainer line of red beet, aiming to solve the technical problem that monoembryonic red beet varieties cannot be cultivated in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is to provide a method for creating a single embryo sterile line and a maintainer line of red beet, comprising:

[0006] Step 1: Year 1: Sowing monoembryonic red beet varieties and polyembryonic red beet lines, extracting leaf genomic DNA from the monoembryonic red beet varieties and polyembryonic red beet lines and performing molecular marker identification, screening the monoembryonic red beet varieties and polyembryonic red beet lines identified by molecular markers, harvesting the mother roots of the screened monoembryonic red beet varieties and polyembryonic red beet lines and performing vernalization;

[0007] Step 2: Second year: Plant the mother roots of the monoembryonic red beet variety and the mother roots of the polyembryonic red beet line harvested in the first year in pairs in the quarantine area;

[0008] The polyembryonic red beet line was pollinated with a monoembryonic red beet variety during the flowering period, and the seeds of the polyembryonic red beet line were harvested and named F 1杂 , for the harvest of F 1杂 The seeds are propagated southward and the mother roots are harvested and placed in the beet cellar for vernalization;

[0009] The monoembryonic red beet variety was self-pollinated and the harvested seeds were named S 1品 , S 1品 Store the seeds in the freezer;

[0010] Step 3: Year 3: Planting seeds of polyembryonic red beet varieties harvested in the second year F 1杂 Mother root, get F 1杂 Single plant, F 1杂 The individual plants were self-pollinated and the individual plant seeds F were harvested. 2杂 , for harvested single plant buckle cover seeds F 2杂 Carry out southern propagation of mother roots, harvest the mother roots and place them in the beet cellar for vernalization;

[0011] For the seeds of monoembryonic red beet varieties harvested in the second year, 1品 Carry out southern propagation to cultivate mother roots, harvest the mother roots and place them in the beet cellar for vernalization;

[0012] Step 4: Year 4: Plant the third year harvested F in the quarantine area 2杂 Seed mother root, get F 2杂 Plants, in F 2杂 At the budding stage, all polyembryonic plants were removed and single embryonic plants were retained to obtain F 2杂 Monoembryonic plants were identified using molecular marker technology. 2杂 The monoembryonic plant nuclear fertility-related gene is retained as a homozygous recessive individual, and the retained homozygous recessive individual is named N-1. The homozygous recessive individual N-1 is self-pollinated, and the seeds of N-1 after self-pollination are harvested and then propagated in the south to cultivate mother roots, which are then placed in a sugar beet cellar for vernalization;

[0013] Planting S. 1品Single plant, in S 1品 During the budding stage of a single plant, all polyembryonic plants were removed and single embryonic plants were retained to obtain S 1品 Monoembryonic plants, the retained S 1品 Monoembryonic plants were surveyed during the flowering period, and plants without pollen were retained, i.e., individuals with homozygous recessive nuclear fertility genes. The retained homozygous recessive individuals were named S-1, and the homozygous recessive individuals S-1 were pollinated by N-1. The pollinated S-1 seeds were harvested and then propagated in the south to cultivate mother roots, which were then placed in a sugar beet cellar for vernalization.

[0014] Step 5: Year 5 to Year 8: Plant the N-1 mother root plants and S-1 mother root plants after vernalization in the fourth year in pairs and treat them to obtain multiple pairs of red beet monoembryonic sterile lines and maintainer lines.

[0015] Optionally, the monoembryonic red beet variety and polyembryonic red beet line screened in step 1 are a monoembryonic red beet variety with a genotype of SMm(Xx) and a polyembryonic red beet line with a genotype of NMM(XX).

[0016] Optional, F in step 2 1杂 The genotypes of the seeds are NMM(XX), NMM(Xx), NMm(XX) and NMm(Xx), S 1品 The genotypes of the seeds are [S(MM, Mm, mm)(Xx, XX, xx)], with a total of nine combinations.

[0017] Optional, F in step 3 2杂 The genotypes of the seeds are NMM(XX), NMM(Xx), NMM(xx), NMm(XX), Nmm(XX), NMm(Xx), NMm(xx), Nmm(Xx) and Nmm(xx).

[0018] Optionally, the treatment of the N-1 mother root plant and the S-1 mother root plant in step 5 includes: continuously self-pollinating the N-1 plant while continuously backcrossing the S-1 plant, amplifying the seeds harvested each year using InDel primers, and releasing them when the nuclear genes reach the homozygous standard to obtain multiple pairs of red beet single embryo sterile lines and maintainer lines.

[0019] The beneficial effects of the method and system for creating a single-embryonic sterile line and a maintainer line of red beet provided by the present invention are as follows: the method for creating a single-embryonic sterile line and a maintainer line of red beet provided by the present invention can increase the number of single-embryonic cytoplasmic male sterile lines and maintainer lines of red beet in my country from zero to a large number, and the single-embryonic rate of the single-embryonic red beet maintainer line seeds reaches more than 98%, the single-embryonic rate of the sterile line is more than 98% and the sterility rate is 100%, providing support for the large-scale creation of single-embryonic red beet single-embryonic cytoplasmic male sterile lines and maintainer lines and the creation of single-embryonic red beet varieties in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 A flow chart of a method for creating a single-embryonic sterile line and a maintainer line of red sugar beet provided in an embodiment of the present invention;

[0022] Figure 2 The band result diagram of the amplification of the genome of the remaining monoembryonic plant leaves using the S17 primer provided in the embodiment of the present invention;

[0023] Figure 3 A diagram showing the results of amplification using S17 primers provided in an embodiment of the present invention;

[0024] Figure 4 The enzyme digestion result diagram provided in the embodiment of the present invention;

[0025] Figure 5 Amplification results of primer ND31 provided in an embodiment of the present invention;

[0026] In the figure, m represents monoembryony, M represents polyembryony, N represents cytoplasmic fertility, S represents cytoplasmic sterility, X represents nuclear fertility, and x represents nuclear sterility. Example

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] like Figure 1 As shown, in the first year: a monoembryonic red beet variety introduced from abroad was selected and its fertility genotype was identified using primers TR1 and S17 related to identifying sugar beet fertility. The embryonic and fertility genotype was [MmS(Xx)] and named A; and a domestic red beet polyembryonic restorer line B was selected, with the embryonic and fertility genotype [MMN(XX)]. A and B were sown in summer, and at least 50 mother roots of each were harvested and stored for vernalization. (Where m: monoembryonic; M: polyembryonic; N: cytoplasmic fertile; S: cytoplasmic sterile; X: nuclear fertile; x: nuclear sterile)

[0029] In the second year, choose an isolated space to plant the mother roots harvested in the first year. Plant A in one row and plant B and A next to each other in another row. While A is self-pollinating, pollinate B and harvest the seeds S on A. 1品 [(MM, Mm, mm)S(XX, Xx, xx)]; and the seed F on B 1杂 [(MM, Mm)N(XX, Xx)]. S 1品 Store the seeds in the freezer. 1杂 The seeds are sown in summer, and at least 100 mother roots are harvested, which are then placed in a cellar for vernalization.

[0030] S 1品 The genotypes of the seeds are SMM(XX), SMM(Xx), SMM(xx), SMm(XX), SMm(Xx), SMm(xx), Smm(XX), Smm(Xx) and Smm(xx).

[0031] Year 3: Harvest F in the second year of planting 1杂 The seed mother root is self-pollinated, and the F 2杂 The seed genotype is [(MM, Mm, mm)N(XX, Xx, xx)]; the F 2杂 The seeds are sown in summer, and the S stored in the refrigerator last year are sown in summer. 1品 seed.

[0032] F 2杂 The genotypes of the seeds are NMM(XX), NMM(Xx), NMM(xx), NMm(XX), Nmm(XX), NMm(Xx), NMm(xx), Nmm(Xx) and Nmm(xx).

[0033] Year 4: Planting the third year harvest F 2杂 Seeds and S 1品 Seeds, flowering eliminated F 2杂 Of the polyembryonic plants, only monoembryonic plants were retained;

[0034] like Figure 2 As shown: The S17 primer was used to amplify the genome of the remaining monoembryonic plants leaves, and the plants with amplified bands of 1800 bp were selected. The amplification results are shown as follows: Figure 3 Then it is digested with HapII and HindIII, according to Figure 4 As shown in the figure, the enzyme digestion products of 1000bp and 700bp were retained, and the rest of the plants were eliminated. The remaining plant N-1 had the gene of the single embryo maintenance line; at the same time, the S 1品The plants are identified and polyembryonic plants are eliminated. The pollen fertility of the retained monoembryonic plants is investigated during the flowering period, and the plants with completely aborted pollen are selected. The retained plant S-1 is the monoembryonic red beet sterile line; then the retained N-1 is used to pollinate S-1, and N-1 is self-pollinated at the same time. After the seeds are harvested, the mother roots for southern propagation are placed in the beet cellar for vernalization.

[0035] Year 5 to Year 8: Plant the N-1 seed mother roots and S-1 seed mother roots harvested in the fourth year to obtain N-1 plants and S-1 plants. Self-pollinate the N-1 plants continuously. Backcross the S-1 plants continuously while self-pollinating the N-1 plants continuously. Figure 5 As shown, InDel primers are used to determine whether the lineage standard is met. After meeting the standard, plants with less than or equal to 2 differential sites are retained, that is, the sterile line and the maintainer line are homozygous, and they can be released to obtain multiple pairs of red beet single embryo sterile lines and maintainer lines. The obtained single embryo rate of the monoembryonic red beet maintainer line seeds is more than 98%, the single embryo rate of the sterile line is more than 98% and the sterility rate is 100%.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for creating a monoembryonic sterile line and a maintainer line of red sugar beet, characterized in that: The method for creating a red beet monoembryonic sterile line and a maintainer line is achieved by the following steps: Step 1: In the first year, monoembryonic red beet varieties and polyembryonic red beet lines are sown, genomic DNA of leaves of the monoembryonic red beet varieties and polyembryonic red beet lines are extracted and molecular markers are identified, the monoembryonic red beet varieties and polyembryonic red beet lines identified with molecular markers are screened, and the mother roots of the screened monoembryonic red beet varieties and polyembryonic red beet lines are harvested and vernalized; Step 2: Second year: Plant the mother roots of the monoembryonic red beet variety and the mother roots of the polyembryonic red beet line harvested in the first year in pairs in the quarantine area; The polyembryonic red beet line was pollinated with a monoembryonic red beet variety during the flowering period, and the seeds of the polyembryonic red beet line were harvested and named F 1杂 , for the harvest of F 1杂 The seeds are propagated southward and the mother roots are harvested and placed in the beet cellar for vernalization; The monoembryo red beet variety was self-pollinated, and the harvested monoembryo red beet variety seeds were named S 1品 , S 1品 Store the seeds in the freezer; Step 3: Year 3: Planting seeds of polyembryonic red beet varieties harvested in the second year F 1杂 Mother root, get F 1杂 Single plant, for the F 1杂 The individual plants were self-pollinated and the individual plant seeds F were harvested. 2杂 , for the harvested single plant buckle cover seeds F 2杂 Carry out southern propagation of mother roots, harvest the mother roots and place them in the beet cellar for vernalization; For the seeds of monoembryonic red beet varieties harvested in the second year, 1品 Carry out southern propagation to cultivate mother roots, harvest the mother roots and place them in the beet cellar for vernalization; Step 4: Year 4: Plant the third year harvested F in the quarantine area 2杂 Seed mother root, get F 2杂 Plants, in the F 2杂 At the budding stage, all polyembryonic plants were removed and single embryonic plants were retained to obtain F 2杂 Monoembryonic plants were identified by molecular marker technology. 2杂 The monoembryonic plant nuclear fertility-related gene is retained as a homozygous recessive individual, and the retained homozygous recessive individual is named N-1. The homozygous recessive individual N-1 is self-pollinated, and the seeds of N-1 after self-pollination are harvested and then propagated in the south to cultivate mother roots, which are then placed in a sugar beet cellar for vernalization; Planting S. 1品 In the S 1品 During the budding stage of a single plant, all polyembryonic plants were removed and single embryonic plants were retained to obtain S 1品 Monoembryonic plants, the retained S 1品 Monoembryonic plants were surveyed during the flowering period, and plants without pollen were retained, i.e., individuals with homozygous recessive nuclear fertility genes. The retained homozygous recessive individuals were named S-1, and the homozygous recessive individuals S-1 were pollinated by N-1. The pollinated S-1 seeds were harvested and then propagated in the south to cultivate mother roots, which were then placed in a sugar beet cellar for vernalization. Step 5: Years 5 to 8: Plant the N-1 mother root plants and S-1 mother root plants after vernalization in the fourth year in pairs and treat them to obtain multiple pairs of red beet monoembryonic sterile lines and maintainer lines; The treatment of the N-1 mother root plant and the S-1 mother root plant in step 5 includes: continuously self-pollinating the N-1 plant and continuously backcrossing the S-1 plant, amplifying the seeds harvested each year using InDel primers, and releasing them when the cell nuclear genes reach the homozygous standard to obtain multiple pairs of red beet monoembryonic sterile lines and maintainer lines.

2. The method for creating a single-embryonic sterile line and maintainer line of red beet according to claim 1, characterized in that: The monoembryonic red beet variety and the polyembryonic red beet line screened in step 1 are a monoembryonic red beet variety with a genotype of SMm (Xx) and a polyembryonic red beet line with a genotype of NMM (XX).

3. The method for creating a single embryo sterile line and maintainer line of red beet according to claim 2, characterized in that: In step 2, F 1杂 The genotypes of the seeds are NMM(XX), NMM(Xx), NMm(XX) and NMm(Xx), S 1品 The genotypes of the seeds are [S(MM, Mm, mm)(Xx, XX, xx)], with a total of nine combinations.

4. The method for creating a single-embryonic sterile line and maintainer line of red beet according to claim 3, characterized in that: In step 3, F 2杂 The genotypes of the seeds are NMM(XX), NMM(Xx), NMM(xx), NMm(XX), Nmm(XX), NMm(Xx), NMm(xx), Nmm(Xx) and Nmm(xx).

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