Method for improving dominant genic male sterility homozygous two-type line of brassica napus
Through molecular marker-assisted breeding methods, the dominant nuclear male sterile lines of Brassica napus were hybridized and backcrossed to screen homozygous sterile plants, which solved the problem of the large workload of backcrossing and genotype identification in Brassica napus breeding, and achieved efficient breeding and stable seed morphology preservation.
Patent Information
- Application Number
- CN202510973390.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-09-26
AI Technical Summary
The existing backcrossing and genotype identification work in Brassica napus breeding is heavy, and homozygous sterile lines need to be preserved through test tube seedlings, which carries uncertainty risks.
Molecular marker-assisted breeding methods are used to cultivate two homozygous dominant nuclear male sterile lines of Brassica napus through hybridization and backcrossing. Molecular markers are used to screen homozygous sterile plants, which are directly preserved in the form of seeds, reducing the workload of backcrossing and genotype identification.
It effectively reduces the breeding workload, improves breeding efficiency, avoids the uncertainty risk of test tube seedling preservation, and directly obtains homozygous two-type lines.
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Figure CN120694159A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of Brassica napus breeding, and in particular to a method for improving a dominant nuclear sterile homozygous two-type line of Brassica napus. Background Art
[0002] As an important oil crop in the world, the utilization of heterosis of Brassica napus is an effective way to improve its yield and quality and enhance its stress resistance. c , sterility gene Ms5 b , the gene Ms5 that restores the sterility gene to fertility a , the explicit and implicit relationship among the three is Ms5 a >Ms5 b >Ms5 c ) controlled genetic model, using a homozygous two-type line, a temporary protection line and a restorer line to produce rapeseed hybrid seeds. In the seed production process, the sterile plants (genotype Ms5) in the homozygous two-type line are first selected. b Ms5 b ) and Linbao line (genotype Ms5 c Ms5 c ) hybridization to produce a fully sterile line, and the following year the fully sterile line was used as the female parent and the restorer line (genotype Ms5 a Ms5 a ) is the male parent to produce hybrid seeds under natural isolation conditions.
[0003] Generally, in breeding, the homozygous two-type lines used to cultivate hybrids are mainly obtained through the following steps:
[0004] (1) Breeding of heterozygous two-type lines with near-isogenic characteristics to the recurrent parent: using the existing dominant genic male sterile lines (Ms5 b Ms5 c ) as the female parent, and the excellent variety (Ms5 c Ms5 c , and used as the recurrent parent) by hybridization and continuous backcrossing to cultivate a heterozygous two-type line (Ms5 b Ms5 c +Ms5 c Ms5 c ).
[0005] (2) Selection of restorer lines with near-isogenic characteristics to the recurrent parent: Based on the existing dominant nuclear sterile restorer line (Ms5 a Ms5 a ) as the female parent, and hybridized and backcrossed with the recurrent parent described in (1). In the backcross process, the homozygous sterile plant (Ms5 b Ms5 b) as the female parent, and perform test cross on the backcross progeny. Select the backcross progeny corresponding to the testcross combination with fertility separation, and continue backcrossing with the recurrent parent. After backcrossing 3 times, select the backcross progeny corresponding to the testcross combination with fertility separation for self-pollination and test cross, and cultivate the restorer line (Ms5) that is nearly isogenic with the recurrent parent described in (1). a Ms5 a ).
[0006] (3) Selection of homozygous two-type lines: Select the sterile plants (Ms5 b Ms5 c ) and (2) the restored line (Ms5 a Ms5 a ) and then from its F1 population (Ms5 a Ms5 b +Ms5 a Ms5 c ) and then select sterile plants and fertile plants from the self-pollination group with fertility separation for brother-sister crosses. The brother-sister cross combination with fertility separation is the homozygous two-type line (Ms5 a Ms5 b +Ms5 b Ms5 b ).
[0007] The existing technology has the following disadvantages: the workload of backcross breeding is large, that is, the gene Ms5 must be used separately. a and Ms5 b Backcross breeding is carried out for the target gene. Genotype identification is a large workload, that is, the gene Ms5 a When backcrossing is carried out for the purpose of breeding, the backcross offspring must be tested with homozygous sterile plants to screen for the allele Ms5. a Individual. Ms5 a and Ms5 b After the backcross breeding is completed, the two near-isogenic lines should be hybridized again to integrate Ms5 a and Ms5 b After integrating into the same group, self-pollination and brother-sister cross-pollination are used to select homozygous two-type lines.
[0008] In order to overcome the above shortcomings, the present invention uses the existing homozygous sterile line as Ms5 b Donor, and superior varieties (Ms5 c Ms5 c , recurrent parent) hybridization and continuous backcrossing to cultivate heterozygous two-type lines (Ms5 b Ms5 c +Ms5 c Ms5 c); when the sterile plants in the heterozygous two-type line first bloom (Ms5 b Ms5 c If there are individual fertile stamens, self-pollinate them through assisted pollination and bagging, and then use the seeds obtained from self-pollination to establish test tube plantlets; with the help of dominant nuclear sterile molecular marker-assisted selection, retain the homozygous sterile line (Ms5) that is nearly isogenic to the recurrent parent. b Ms5 b ); then the excellent recovery line (Ms5 a Ms5 a ) is Ms5 a The donor was hybridized and backcrossed with the newly bred homozygous sterile line to obtain a homozygous two-type line (Ms5 b Ms5 b +Ms5 a Ms5 b ). Summary of the Invention
[0009] The present invention provides a method for directly improving the dominant genic male sterile homozygous two-type line of Brassica napus using molecular marker-assisted breeding, which effectively reduces the workload of backcross breeding and genotype identification and can greatly improve breeding efficiency. In addition, the obtained homozygous two-type line (Ms5 b Ms5 b +Ms5 a Ms5 b ) is preserved in seed form, and there is no need to preserve the homozygous sterile line (Ms5 b Ms5 b ) to reduce the uncertainty risk during the storage of test tube seedlings. The specific steps are as follows:
[0010] Step 1.1: Breeding of heterozygous two-type lines that are nearly isogenic to the recurrent parent:
[0011] Step 1.1.1: Use the existing dominant nuclear sterile homozygous clone (Ms5 b Ms5 b ) is the female parent, the excellent variety (Ms5 c Ms5 c , recurrent parent) is the male parent of the hybrid, and the F1 population is all sterile, with the genotype of Ms5 b Ms5 c .
[0012] Step 1.1.2: The F1 population described in step 1.1.1 is used as the female parent and backcrossed with the recurrent parent. The sterile and fertile plants in the BC1 population each account for 50%, and the genotypes are Ms5 b Ms5 c and Ms5 c Ms5 cAccording to this method, three backcrosses were carried out to cultivate a heterozygous two-type line with nearly isogenic genes to the recurrent parent, with sterile plants and fertile plants accounting for 50% each. The genotypes were Ms5 b Ms5 c and Ms5 c Ms5 c .
[0013] Step 1.2 Establishment of homozygous sterile plantlets in vitro:
[0014] Step 1.2.1: The sterile plants (Ms5 b Ms5 c ) as the material, using a very small amount of pollen from individual stamens at the time of initial flowering to supplement pollination of other flowers on the sterile plant, and then bagging and selfing. After maturity, the bagged and selfed siliques were harvested, threshed, and the seeds collected.
[0015] Step 1.2.2: The seeds described in step 1.2.1 were sterilized and placed on B5 medium. After emergence, test tube seedlings were established according to the individual plants. The seedlings were numbered and a small piece of leaf was taken for DNA extraction. The DNA was amplified using the molecular marker BE10. The number of the amplified band with only 305 bp was selected, which was the homozygous sterile plant (genotype Ms5). b Ms5 b ), retain the corresponding test tube seedlings for propagation.
[0016] Step 1.3: Breeding of homozygous two-type lines that are nearly isogenic to the recurrent parent:
[0017] Step 1.3.1: Use the above-mentioned preserved test tube seedlings as the female parent and the excellent restorer line (Ms5 a Ms5 a ) is the male parent hybrid, the F1 population is all fertile, and the genotype is Ms5 a Ms5 b .
[0018] In step 1.3.2, the above-retained test tube seedlings were used as the female parent, and the F1 population described in step 1.3.1 was used as the male parent for backcrossing. In the BC1 population, the sterile plants and fertile plants each accounted for 50%, and the genotypes were Ms5 b Ms5 b and Ms5 a Ms5 b .
[0019] In step 1.3.3, the above-retained test tube seedlings are used as the female parent, and the fertile plants in the BC1 population described in step 1.3.2 are backcrossed to the male parent. The sterile plants and fertile plants in the BC2 population each account for 50%, and the genotypes are Ms5 b Ms5 b and Ms5 a Ms5 b, and then two more backcrosses were made according to this method to cultivate the BC4 population that is nearly isogenic to the recurrent parent, with 50% fertile plants and 50% sterile plants, and the genotypes are Ms5 a Ms5 b and Ms5 b Ms5 b , this population is a homozygous two-type line that is nearly isogenic to the recurrent parent.
[0020] The present invention has the following advantages: (1) No need to use Ms5 a To carry out backcross breeding for the target gene, there is no need to perform Ms5 on the backcross offspring. a Allele test cross identification. (2) No need to preserve homozygous sterile lines (Ms5 b Ms5 b ), but rather preserve the homozygous two-type line (Ms5 b Ms5 b +Ms5 a Ms5 b ). BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The gene Ms5 b Schematic diagram of the target backcross breeding and the heterozygous two-type line with near-isogenic characteristics of the recurrent parent.
[0022] Figure 2 The gene Ms5 a Schematic diagram of the target backcross breeding of a restorer line that is nearly isogenic to the recurrent parent.
[0023] Figure 3 Is the gene Ms5 a and Ms5 b Schematic diagram of screening homozygous sterile lines by integrating them into the same progeny population.
[0024] Figure 4 This is a schematic diagram of the improvement method of the dominant genic male sterile homozygous two-type line of Brassica napus. DETAILED DESCRIPTION
[0025] The present invention will be described in further detail below with reference to the accompanying drawings.
[0026] In conjunction with all the accompanying drawings, a method for improving a dominant genic male sterile homozygous two-type line of Brassica napus comprises the following steps:
[0027] Step 1.1 Gene Ms5 b The goal is to backcross and breed a two-type heterozygous line that is nearly isogenic to the recurrent parent:
[0028] Step 1.1.1: Use the existing dominant nuclear sterile homozygous clone (Ms5 bMs5 b ) is the female parent, the excellent variety (Ms5 c Ms5 c , recurrent parent) is the male parent of the hybrid, and the F1 population is all sterile, with the genotype of Ms5 b Ms5 c .
[0029] Step 1.1.2: The F1 population described in step 1.1.1 is used as the female parent and backcrossed with the recurrent parent. The sterile and fertile plants in the BC1 population each account for 50%, and the genotypes are Ms5 b Ms5 c and Ms5 c Ms5 c . According to this method, three backcrosses were performed continuously to cultivate a heterozygous two-type line that was nearly isogenic to the recurrent parent.
[0030] The sterile and fertile plants in the heterozygous two-type line cultivated above, which are nearly isogenic to the recurrent parent, account for 50% each and have genotypes of Ms5 b Ms5 c and Ms5 c Ms5 c ;
[0031] Step 1.2 Establishment of homozygous sterile plantlets in vitro:
[0032] Step 1.2.1: The sterile plants (Ms5 b Ms5 c ) as the material, using a very small amount of pollen from individual stamens at the time of initial flowering to supplement pollination of other flowers on the sterile plant, and then bagging and selfing. After maturity, the bagged and selfed siliques were harvested, threshed, and the seeds collected.
[0033] Step 1.2.2: The seeds described in step 1.2.1 are sterilized and placed on B5 culture medium. After emergence, test tube seedlings are established according to individual plants. A small piece of leaf is numbered and taken for DNA extraction and amplification using molecular marker BE10.
[0034] In step 1.2.3, the PCR amplification products were separated by 2% agarose gel electrophoresis. Three amplification band types were found: one amplification band of 398 bp; one amplification band of 305 bp; and two amplification bands of 305 bp + 398 bp.
[0035] Step 1.2.4: From the electrophoretogram obtained in step 1.2.3, select the number of the amplified band with only 305 bp, i.e., the homozygous sterile strain (genotype Ms5 b Ms5 b ), retain the corresponding test tube seedlings for propagation.
[0036] Step 1.3: Breeding of homozygous two-type lines that are nearly isogenic to the recurrent parent:
[0037] Step 1.3.1: The test tube seedlings obtained in step 1.2.4 were used as the female parent, and the excellent restorer line (Ms5 a Ms5 a ) is the male parent hybrid, the F1 population is all fertile, and the genotype is Ms5 a Ms5 b .
[0038] In step 1.3.2, the test tube seedlings obtained by screening in step 1.2.4 are used as the female parent, and the F1 population in step 1.3.1 is backcrossed to the male parent. The sterile and fertile plants in the BC1 population each account for 50%, and the genotypes are Ms5 b Ms5 b and Ms5 a Ms5 b .
[0039] In step 1.3.3, the test tube seedlings obtained by screening in step 1.2.4 are used as the female parent, and the fertile plants in the BC1 population in step 1.3.2 are backcrossed to the male parent. The sterile plants and fertile plants in the BC2 population each account for 50%, and the genotypes are Ms5 b Ms5 b and Ms5 a Ms5 b , and then two more backcrosses were made according to this method to cultivate the BC4 population that is nearly isogenic to the recurrent parent, with 50% fertile plants and 50% sterile plants, and the genotypes are Ms5 a Ms5 b and Ms5 b Ms5 b This BC4 population is a homozygous two-type line (Ms5 b Ms5 b +Ms5 a Ms5 b ).
[0040] In the specific implementation of the present invention, the existing homozygous sterile line (Ms5 b Ms5 b ) is Ms5 b Donor, and superior varieties (Ms5 c Ms5 c , and used as the recurrent parent) were hybridized and backcrossed to obtain a heterozygous two-type line that was nearly isogenic to the recurrent parent; at the initial flowering stage, the sterile plants (Ms5 b Ms5 c ) were self-pollinated and established into test tube seedlings. Through molecular marker-assisted selection, homozygous sterile lines (Ms5) with near-isogenic characteristics to the recurrent parent were selected. bMs5 b ); then the excellent recovery line (Ms5 a Ms5 a ) is Ms5 a The donor was hybridized and backcrossed with the newly bred homozygous sterile line to obtain a homozygous two-type line (Ms5 b Ms5 b +Ms5 a Ms5 b ).
[0041] Prior art: Reference Figure 1-2 , must target gene Ms5 b and Ms5 a Backcrossing should be carried out to obtain heterozygous two-type lines and restorer lines that are nearly isogenic to the recurrent parent. In addition, the restorer lines that are nearly isogenic to the recurrent parent must be tested with homozygous sterile plants to identify the carriers of Ms5. a The workload of backcross breeding and genotype identification is large. Figure 3 , and finally Figure 1 and Figure 2 The two near-isogenic lines obtained must be hybridized and self-pollinated again before the homozygous two-type line can be screened out, which requires a long period of time and a large amount of work.
[0042] The present invention: based on the existing dominant nuclear sterile homozygous sterile line (Ms5 b Ms5 b ) is the female parent, the excellent variety (Ms5 c Ms5 c , recurrent parent) as the male parent, through hybridization and backcrossing, a heterozygous two-type line with nearly isogenic genes to the recurrent parent is bred. Then, the sterile plants in the newly bred heterozygous two-type line are first floridated (Ms5 b Ms5 c If there are individual fertile stamens, self-pollination is performed through assisted pollination and bagging, followed by molecular marker-assisted selection to retain the homozygous sterile line that is nearly isogenic to the recurrent parent and to combine it with the superior restorer line (Ms5 a Ms5 a ) hybridization to obtain the F1 population, and then use the F1 population as the male parent and the above homozygous sterile line as the recurrent parent, and backcross 3-4 times continuously to obtain a homozygous two-type line (Ms5) that is nearly isogenic to the recurrent parent. a Ms5 b and Ms5 b Ms5 b ).
[0043] Embodiments of the present invention:
[0044] During the rapeseed flowering period in 2015, the dominant genic male sterile homozygous clone 15CV03 was used as the female parent and the double-low rapeseed variety 'Zheyou 50' was used as the male parent to obtain F1 generation seeds.
[0045] During the flowering period in 2016, all F1 generation (field number 18-F001) were sterile, and backcrossing was carried out with it as the female parent and 'Zheyou 50' as the recurrent parent.
[0046] During the flowering period in 2017, the sterile plant in the BC1 population ‘17-BC103’ was the female parent, and ‘Zheyou 50’ was the recurrent parent for backcrossing.
[0047] In 2018, a backcross was conducted using the sterile plant in the BC2 population '18-BC205' as the female parent and 'Zheyou 50' as the recurrent parent.
[0048] In 2019, the fertility of the BC3 population '19-BC301' was segregated. A single plant numbered 19-BC301-7 had a very small amount of pollen on the stamens of the first few flowers at the time of initial flowering. This pollen was used to assist in pollination of other flowers on the plant and self-pollinated in bags. After bagging, 53 BC3F2 seeds were obtained from the sterile plant. These seeds were used as donors to establish test tube plantlets (numbered 19CV07). The molecular marker BE10 was then used to identify the genotype of these test tube plantlets. After identification, only a 305bp amplification band was found in 13 test tube plantlets, indicating that the genotype of these individual plants was Ms5. b Ms5 b , which are homozygous sterile plants, retain the corresponding test tube seedlings for propagation. In November, some test tube seedlings will be stored in the field.
[0049] In 2020, 20-CV07 was used as the female parent and the dominant nuclear sterile restorer line 'Huyou 3302' was used as the male parent to obtain the F1 population.
[0050] In 2021, all F1 populations 21-CV701 were fertile. Using this as the male parent and 20-CV07 as the female parent, backcrossing was performed to obtain the BC1 population.
[0051] In 2022, fertility segregation was observed in the BC1 population. Backcrossing was continued using the fertile plant as the male parent and 20-CV07 as the female parent to obtain the BC2 population.
[0052] In 2023, fertility segregation was observed in the BC2 population. Backcrossing was continued using the fertile plant as the male parent and 20-CV07 as the female parent to obtain the BC3 population.
[0053] In 2024, fertility segregation was observed in the BC3 population. With the fertile plant as the male parent and 20-CV07 as the female parent, backcrossing was continued to obtain the BC4 population, which was a homozygous two-type line (Ms5) with near-isogenic characteristics to the recurrent parent. b Ms5 b +Ms5 a Ms5b ).
[0054] The above description of the present invention and its embodiments is non-limiting, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by the above description and, without departing from the purpose of the present invention, designs a structure and embodiment similar to the technical solution without creatively designing, they shall fall within the scope of protection of the present invention.
Claims
1. A method for improving a dominant genic male sterile homozygous two-type line in Brassica napus, characterized by: The following steps are involved: Step 1.1 Gene Ms5 b The goal is to backcross and breed a two-type heterozygous line that is nearly isogenic to the recurrent parent: Step 1.1.1: Use the existing dominant nuclear sterile homozygous clone (Ms5 b Ms5 b ) is the female parent, the excellent variety (Ms5 c Ms5 c , recurrent parent) is the male parent of the hybrid, and the F1 population is all sterile, with the genotype of Ms5 b Ms5 c . Step 1.1.2: The F1 population described in step 1.1.1 is used as the female parent and backcrossed with the recurrent parent. The sterile and fertile plants in the BC1 population each account for 50%, and the genotypes are Ms5 b Ms5 c and Ms5 c Ms5 c . According to this method, three backcrosses were performed continuously to cultivate a heterozygous two-type line that was nearly isogenic to the recurrent parent. The sterile and fertile plants in the heterozygous two-type line cultivated above, which are nearly isogenic to the recurrent parent, account for 50% each and have genotypes of Ms5 b Ms5 c and Ms5 c Ms5 c ; Step 1.2 Establishment of homozygous sterile plantlets in vitro: Step 1.2.1: The sterile plants (Ms5 b Ms5 c ) as the material, using a very small amount of pollen from individual stamens at the time of initial flowering to supplement pollination of other flowers on the sterile plant, and then bagging and selfing. After maturity, the bagged and selfed siliques were harvested, threshed, and the seeds collected. Step 1.2.2: The seeds described in step 1.2.1 are sterilized and placed on B5 culture medium. After emergence, test tube seedlings are established according to individual plants. A small piece of leaf is numbered and taken for DNA extraction and amplification using molecular marker BE10. In step 1.2.3, the PCR amplification products were separated by 2% agarose gel electrophoresis. Three amplification band types were found: one amplification band of 398 bp; one amplification band of 305 bp; and two amplification bands of 305 bp + 398 bp. Step 1.2.4: From the electrophoretogram obtained in step 1.2.3, select the number of the amplified band with only 305 bp, i.e., the homozygous sterile strain (genotype Ms5 b Ms5 b ), retain the corresponding test tube seedlings for propagation. Step 1.3: Breeding of homozygous two-type lines that are nearly isogenic to the recurrent parent: Step 1.3.1: The test tube seedlings obtained in step 1.2.4 were used as the female parent, and the excellent restorer line (Ms5 a Ms5 a ) is the male parent hybrid, the F1 population is all fertile, and the genotype is Ms5 a Ms5 b . In step 1.3.2, the test tube seedlings obtained by screening in step 1.2.4 are used as the female parent, and the F1 population in step 1.3.1 is backcrossed to the male parent. The sterile and fertile plants in the BC1 population each account for 50%, and the genotypes are Ms5 b Ms5 b and Ms5 a Ms5 b . In step 1.3.3, the test tube seedlings obtained by screening in step 1.2.4 are used as the female parent, and the fertile plants in the BC1 population in step 1.3.2 are backcrossed to the male parent. The sterile plants and fertile plants in the BC2 population each account for 50%, and the genotypes are Ms5 b Ms5 b and Ms5 a Ms5 b , and then two more backcrosses were made according to this method to cultivate the BC4 population that is nearly isogenic to the recurrent parent, with 50% fertile plants and 50% sterile plants, and the genotypes are Ms5 a Ms5 b and Ms5 b Ms5 b This BC4 population is a homozygous two-type line (Ms5 b Ms5 b +Ms5 a Ms5 b ).