A rapid conversion method for radish sclerotium-plasma interaction male sterility lines
By introducing sterility genes into superior radish varieties and using generation technology, the problem of long conversion time for male sterile lines with nucleocytoplasmic interaction in superior radish varieties has been solved. Conversion can be completed within 10 years, and the effects of gene linkage can be reduced. The resulting sterile lines are closer to the materials to be converted.
Patent Information
- Application Number
- CN202410087535.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-22
- Publication Date
- 2025-10-28
- Estimated Expiration
- 2044-01-22
AI Technical Summary
Existing technologies for converting superior radish varieties into nucleocytoplasmic male-sterile lines that do not contain sterility genes in the cell nucleus require excessively long conversion times, typically more than 12 years, and the resulting sterile lines may contain other genes linked to the sterility gene.
The sterility gene was introduced by crossing existing nucleocytoplasmic interaction maintainer lines with superior radish varieties. By rationally controlling the number of seed plants retained in each generation and combining generation technology, two generations of segregation and test cross were carried out in the second stage to shorten the breeding time. Semi-sterile lines were selected to reduce the influence of gene linkage.
The conversion time was shortened to 10 years, reducing the workload, and the male-sterile lines developed were closer to the materials to be converted, reducing the impact of gene linkage.
Abstract
Description
Technical Field
[0001] This invention relates to the field of male sterility line generation conversion technology, specifically to a rapid conversion method for radish core-cytoplasmic interaction male sterility lines. Background Technology
[0002] Radishes are a type of cruciferous vegetable, suitable for fresh consumption, cooked consumption, and processing, and are widely cultivated in China. Currently, the varieties used in production are mainly hybrids. There are two methods for creating hybrids: using self-incompatible lines and using male-sterile lines for testing. Hybrids created using self-incompatible lines have slightly lower uniformity and are more prone to parental loss; while hybrids created using male-sterile lines have better uniformity and are less likely to lose parental lines. Therefore, using male-sterile lines to create hybrids is the most common method in radish hybrid production, and the conversion of male-sterile lines is a major task in radish breeding.
[0003] Male sterility commonly includes three types: nuclear sterility, cytoplasmic sterility, and nucleocytoplasmic interaction sterility. Nuclear sterility involves the presence of the sterility gene only in the cell nucleus, generally yielding only 50% sterile lines, making it difficult to utilize in production. Cytoplasmic sterility involves the presence of the sterility gene only in the cytoplasm; currently, most cytoplasmic sterility cases suffer from yellowing, resulting in low seed production. Nucleocytoplasmic interaction sterility involves the presence of the sterility gene in both the cytoplasm and the nucleus, allowing for the selection of stable maintainer lines (containing fertile genes in the cytoplasm and homozygous sterile genes in the nucleus; the offspring of self-crossing are maintainer lines) and sterile lines (containing sterile genes in the cytoplasm and homozygous sterile genes in the nucleus; the offspring of crossing with the maintainer line are sterile lines). Nucleocytoplasmic interaction sterility can produce stable 100% sterile lines and is the most commonly used method in breeding.
[0004] However, some superior radish varieties lack the ability to maintain the sterile source, meaning their cell nuclei do not contain the corresponding sterility gene. Therefore, the common method of selecting sterile lines through paired testcrosses cannot be used. In this case, the existing method for converting sterile lines involves crossing the radish variety to be converted with the sterile source to obtain a hybrid. This hybrid is then used as the male parent and crossed with the radish variety to be converted to introduce the sterility gene. Finally, maintainer lines and sterile lines are selected through backcrossing, testcrossing, and self-pollination. Because backcrossing and testcrossing require too many generations, and root morphology selection can only be performed once a year, this method typically takes more than 12 years to complete the conversion. Summary of the Invention
[0005] This invention discloses a rapid conversion method for nucleocytoplasmic male sterile lines in radishes. The technical problem this method aims to solve is to shorten the conversion time for superior radish varieties with nucleocytoplasmic male sterile lines that do not contain sterility genes in their cell nuclei, and to develop male sterile lines that are closer to the superior radish varieties. When the superior radish varieties do not contain the corresponding sterility gene in their cell nuclei, the method involves introducing the sterility gene through hybridization between existing nucleocytoplasmic interaction maintainer lines and superior radish varieties; rapidly increasing the number of generations by using generation-increasing techniques while reasonably controlling the number of seed plants retained in each generation; and identifying and selecting root morphology at appropriate generations to convert the male sterile lines. This method reduces the number of generations by one compared to conventional methods, shortening the development time to 10 years, reducing workload, and producing male sterile lines that are closer to the materials to be converted.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A rapid conversion method for male sterile lines of radish with cytoplasmic interaction is proposed. Taking Xinlimei radish as an example, the stable inbred line 173-4, which has been selected through multiple generations of self-pollination of Xinlimei radish, is selected as the recurrent parent for conversion of Xinlimei male sterile lines. The male sterile line W9A of green radish with a sterility rate of 100% is selected, and the maintainer line W9 of green radish is selected as the male parent for conversion. The conversion process includes: backcrossing of superior radish varieties and self-pollination and pair testcrossing of male sterile lines.
[0008] The preferred backcrossing of superior radish varieties includes the following steps:
[0009] (11) In the first year, in the spring, the regenerated parent 173-4 was used as the female parent and the green radish maintainer line W9 was used as the male parent to carry out hybridization to obtain hybrid F1; in the autumn, two single plants were selected from hybrid F1 for self-pollination to obtain self-pollinated seeds S1.
[0010] (12) In the spring of the second year, 30 self-pollinated seeds of S1 single plants were used as male parents and testcrossed with sterile line W9A to obtain testcross S1F. The same 30 self-pollinated seeds of S1 single plants were used as female parents and backcrossed with fertility recurrent parent 173-4 to obtain backcross BC1. In the autumn, the testcross S1F and backcross BC1 obtained in the spring were subjected to low temperature treatment and planted in a winter greenhouse to make them flower. According to fertility, 30 single plants were selected from the backcross BC1 line corresponding to the all-sterile testcross S1F line for self-pollination to obtain SBC1.
[0011] (13) In the spring of the third year, 30 SBC1 single plants were used as male parents and testcrossed with sterile plants in S1F to obtain testcross S2F. The same 30 SBC1 single plants were used as female parents and backcrossed with the fertility recurrent parent 173-4 to obtain backcross BC2. In the autumn, the testcross S2F and backcross BC2 obtained in the spring were treated with low temperature and planted in a winter-warm greenhouse to make them flower. According to fertility, 30 single plants were selected from the BC2 line corresponding to the all-sterile S2F line for self-pollination to obtain SBC2.
[0012] (14) Repeat step (3) in the fourth and fifth years to obtain S4F and SBC4;
[0013] (15) In the sixth spring, 30 SBC4 single plants were used as male parents and testcrossed with sterile plants in S4F to obtain testcross S5F. The same 30 SBC4 single plants were used as female parents and backcrossed with the 173-4 regeneration parent to obtain backcross BC5. In the autumn, the testcross S5F and backcross BC5 obtained in the spring were treated with low temperature and planted in a winter-warm greenhouse to make them flower. According to fertility, 200 single plants were selected from the BC5 lines corresponding to some sterile S5F lines for self-pollination to obtain SBC5.
[0014] Preferably, the self-pollination and sterile line pair testcross portion includes the following steps;
[0015] (21) In the spring of the seventh year, 200 SBC5 plants were self-crossed to obtain S1, and at the same time, S1A was obtained by testcrossing with sterile plants in S5F. In the autumn, S1 and S1A were multiplied, and their marketability, disease resistance and quality were identified by sowing. Based on the autumn sowing identification results and fertility results, some sterile S1A lines and corresponding S1 lines that are similar to the materials to be converted were selected. 100 single plants of S1 line were self-crossed to obtain S2, and at the same time, S2A was obtained by testcrossing with the corresponding S1A.
[0016] (22) Repeat step (5) in the eighth year but do not conduct autumn sowing identification to obtain 100 self-pollinated materials S4 and corresponding S4A;
[0017] (23) In the spring of the ninth year, self-pollination and test crosses of materials continued. In the spring of this year, 100 individual plants of the all-sterile S5A line and the corresponding S5 were selected for self-pollination and test crosses for seed saving. The seed saving quantity of individual plants for self-pollination and test crosses was 150-200 seeds for autumn sowing and identification. In autumn, S5 and S5A were multiplied, and S5 and the corresponding test cross S5A were planted in open field. Based on commerciality, disease resistance and variety, S5 and S5A with similar recurrent parents were selected for self-pollination and test crosses to obtain S6 and S6A. S6 is the maintainer line and S6A is the sterile line.
[0018] (24) In the spring of the tenth year, S6 and S6A were treated with low temperature, sown in the same net and mixed for seed retention, and mixed S6 and S6A were obtained. They were then identified in autumn sowing. Thus, the male sterile line of Xinlimei material 173-4 was successfully bred.
[0019] Preferably, the recurrent parent 173-4 is a stable inbred line bred through 8 generations of self-pollination selection of the Xinlimei radish.
[0020] The beneficial effects of the present invention on the rapid conversion method of radish core-cytoplasm interaction male sterility line:
[0021] Compared with existing technologies, the method of this invention is innovative in three aspects. First, it shortens the generation of breeding and reduces the number of F1 seed plants. Regarding the introduction of the sterile gene into the nucleus, this invention uses an existing maintainer line as the male parent to cross with the material to be bred, thereby introducing the sterile gene. Existing technologies use the material to be bred as the male parent to cross with a sterile source to obtain a hybrid, and then use the hybrid as the male parent to cross with the material to be bred to introduce the sterile gene. Therefore, the method of this invention reduces the generation of breeding by one generation compared to existing technologies. Based on the theory that nucleocytoplasmic male sterility is controlled by two pairs of recessive genes in the nucleus, existing technologies use hybrids as the male parent to introduce the sterile gene, requiring at least four F1 plants to ensure the introduction of the two recessive genes, and in practice, generally around ten plants are needed. Theoretically, the method of this invention only requires one F1 plant to ensure the introduction of the two recessive genes, and in practice, only two plants need to be selected. Second, it shortens the breeding period from 12-14 years to 10 years. The entire conversion process can be divided into two stages: backcrossing of the material to be converted and self-pollination and paired testcrosses of the material to be converted. In the second stage, existing technologies typically involve autumn sowing for identification and selection, with only one generation of segregation and testcrosses per year, resulting in a total conversion period of approximately 12 years. This invention, in the second stage, employs a generation-addition technique under the condition of rationally determining the number of seed plants retained in each generation, allowing for two generations of segregation and testcrosses per year, shortening the entire breeding period to 10 years. Third, it breaks the linkage between the introduced sterility gene and other genes, resulting in a male-sterile line that is more closely similar to the material to be converted. In the second stage of the conversion process, existing technologies generally use completely sterile or highly sterile lines. In this case, the resulting sterile lines will contain some other genes linked to the sterility gene (the sterility source gene linked to the sterility gene when it was introduced). In the first four generations of the second stage of this invention, semi-sterile lines are used. In this case, the resulting sterile lines contain relatively fewer other genes linked to the sterility gene (existing maintainer line genes linked to the sterility gene when it was introduced), and the resulting sterile lines are closer to the material to be converted. This invention utilizes existing maintainer lines to introduce the sterility gene, and by employing a generation-addition technique while reasonably controlling the number of seed plants retained in each generation, it shortens the conversion time of male-sterile lines of superior radish materials that do not contain the sterility gene in their cell nucleus, reduces workload, and accelerates the breeding progress of superior radish hybrids. Detailed Implementation
[0022] The technical solutions of this invention will now be clearly and completely described. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0023] A rapid conversion method for male-sterile lines of radish with sclerotium-cytoplasm interaction, taking the conversion of the Xinlimei radish male-sterile line as an example, includes the following:
[0024] 1. The Xinlimei sterile line, with its recurrent parent 173-4, is a local variety from Liuting Street, Chengyang District, Qingdao. It is a stable inbred line developed through eight generations of self-pollination and selection. Two hundred individual plants from this line were testcrossed with the green radish sterile line Jinhuatai 48A. No sterile plants were found in the testcross combination, confirming that the inbred line does not contain the sterility gene in its cell nucleus. This inbred line has roots approximately 15cm long and 6cm thick, with a green upper and white lower skin, red flesh, and good quality. It is relatively resistant to downy mildew and viral diseases, exhibits mosaic patterns, and has semi-erect leaf clusters.
[0025] 2. The male parent and sterile line are the selected green radish maintainer lines W9 and W9A. This sterile line has a 100% sterility rate, normal flowering, normal nectar glands, small anthers, and no pollen.
[0026] 3. Transplantation process:
[0027] (1) In the first year, in the spring, the regenerated parent 173-4 was used as the female parent and the green radish maintainer line W9 was used as the male parent to carry out hybridization to obtain hybrid F1; in the autumn, two single plants were selected from hybrid F1 for self-pollination to obtain self-pollinated seeds S1.
[0028] (2) In the spring of the second year, 30 self-pollinated seeds of S1 single plants were used as male parents and testcrossed with sterile line W9A to obtain testcross S1F. The same 30 self-pollinated seeds of S1 single plants were used as female parents and backcrossed with fertility recurrent parent 173-4 to obtain backcross BC1. In the autumn, the testcross S1F and backcross BC1 obtained in the spring were subjected to low temperature treatment and planted in a winter greenhouse to make them flower. According to fertility, 30 single plants were selected from the backcross BC1 line corresponding to the all-sterile testcross S1F line for self-pollination to obtain SBC1.
[0029] (3) In the spring of the third year, 30 SBC1 single plants were used as male parents and testcrossed with sterile plants in S1F to obtain testcross S2F. The same 30 SBC1 single plants were used as female parents and backcrossed with the fertility recurrent parent 173-4 to obtain backcross BC2. In the autumn, the testcross S2F and backcross BC2 obtained in the spring were treated with low temperature and planted in a winter-warm greenhouse to make them flower. According to fertility, 30 single plants were selected from the BC2 line corresponding to the all-sterile S2F line for self-pollination to obtain SBC2.
[0030] (4) Repeat step (3) in the fourth and fifth years to obtain S4F and SBC4.
[0031] (5) In the sixth spring, 30 SBC4 single plants were used as male parents and testcrossed with sterile plants in S4F to obtain testcross S5F. The same 30 SBC4 single plants were used as female parents and backcrossed with the fertility recurrent parent 173-4 to obtain backcross BC5. In the autumn, the testcross S5F and backcross BC5 obtained in the spring were treated with low temperature and planted in a winter-warm greenhouse to make them flower. According to fertility, 200 single plants were selected from the BC5 lines corresponding to some sterile S5F lines for self-pollination to obtain SBC5.
[0032] The above describes the backcrossing and breeding stage of superior radish varieties; the following describes the self-pollination and testcrossing stage of male-sterile lines.
[0033] (6) In the spring of the seventh year, 200 SBC5 plants were self-crossed to obtain S1, and at the same time, S1A was obtained by testcrossing with sterile plants in S5F. In the autumn, S1 and S1A were multiplied, and their marketability, disease resistance and quality were identified by sowing. Based on the autumn sowing identification results and fertility results, some sterile S1A lines and corresponding S1 lines that are similar to the materials to be converted were selected. 100 single plants of S1 line were self-crossed to obtain S2, and at the same time, S2A was obtained by testcrossing with the corresponding S1A.
[0034] (7) Repeat step (5) in the eighth year but do not conduct autumn sowing identification to obtain 100 self-pollinated materials S4 and corresponding S4A;
[0035] (8) In the spring of the ninth year, self-pollination and test crosses of materials continued. In the spring of this year, 100 individual plants of the completely sterile S5A line and the corresponding S5 were selected for self-pollination and test crosses for seed saving. The seed saving quantity of individual plants for self-pollination and test crosses was 150-200 seeds for autumn sowing and identification. In autumn, S5 and S5A were multiplied, and S5 and the corresponding test cross S5A were planted in open field. Based on commerciality, disease resistance and variety, S5 and S5A with similar recurrent parents were selected for self-pollination and test crosses to obtain S6 and S6A. S6 is the maintainer line and S6A is the sterile line.
[0036] (9) In the spring of the tenth year, S6 and S6A were treated with low temperature, sown in the same net and mixed for seed retention, and mixed S6 and S6A were obtained. They were then identified in autumn sowing. Thus, the male sterile line of Xinlimei material 173-4 was successfully converted.
[0037] Note regarding the codes used in the text:
[0038] F: hybridization;
[0039] S: self-fertilization;
[0040] BC: backcross;
[0041] A: Sterile line;
[0042] F1: First generation hybrid;
[0043] S1: Self-pollination of F1 generation during the backcrossing and breeding stage; self-pollination of SBC5 during the self-pollination and segregation stage;
[0044] S1F: S1F is a test cross between S1 and a sterile source;
[0045] S2F: Testcross of SBC1 with a sterile source, and so on for the others;
[0046] BC1: BC is the backcross, 1 is the backcross generation, and so on.
[0047] SBC1: Self-crossing after backcrossing 1 generation, and so on for the others;
[0048] S1A: Testcross of S1 and sterile source during the self-pollination separation stage, where 1 is the self-pollination generation, and so on.
Claims
1. A rapid conversion method for radish sclerotium-cytoplasmic interaction male sterility lines, characterized by: Taking the Xinlimei radish as an example, the stable inbred line 173-4, which was bred through multiple generations of self-pollination of Xinlimei radish, was selected as the recurrent parent for the conversion of the Xinlimei sterile line. The sterile line W9A of green radish with a sterility rate of 100% was selected, and the maintainer line W9 of green radish was selected as the male parent for conversion. The conversion process includes: backcrossing of superior radish lines and self-pollination and testcrossing of sterile lines. The backcrossing of superior radish varieties includes the following steps: (11) In the first year, in the spring, the regenerated parent 173-4 was used as the female parent and the green radish maintainer line W9 was used as the male parent to carry out hybridization to obtain hybrid F1; in the autumn, two single plants were selected from hybrid F1 for self-pollination to obtain self-pollinated seeds S1. (12) In the spring of the second year, 30 self-pollinated seeds of S1 single plants were used as male parents and testcrossed with sterile line W9A to obtain testcross S1F. The same 30 self-pollinated seeds of S1 single plants were used as female parents and backcrossed with fertility recurrent parent 173-4 to obtain backcross BC1. In the autumn, the testcross S1F and backcross BC1 obtained in the spring were subjected to low temperature treatment and planted in a winter greenhouse to make them flower. According to fertility, 30 single plants were selected from the backcross BC1 line corresponding to the all-sterile testcross S1F line and self-pollinated to obtain SBC1. (13) In the spring of the third year, 30 SBC1 single plants were used as male parents and testcrossed with sterile plants in S1F to obtain testcross S2F. The same 30 SBC1 single plants were used as female parents and backcrossed with the fertility recurrent parent 173-4 to obtain backcross BC2. In the autumn, the testcross S2F and backcross BC2 obtained in the spring were treated with low temperature and planted in a winter-warm greenhouse to make them flower. According to fertility, 30 single plants were selected from the BC2 line corresponding to the all-sterile S2F line for self-pollination to obtain SBC2. (14) Repeat step (13) in the fourth and fifth years to obtain S4F and SBC4; (15) In the sixth spring, 30 SBC4 single plants were used as male parents and testcrossed with sterile plants in S4F to obtain testcross S5F. The same 30 SBC4 single plants were used as female parents and backcrossed with the fertilization recurrent parent 173-4 to obtain backcross BC5. In the autumn, the testcross S5F and backcross BC5 obtained in the spring were treated with low temperature and planted in a winter-warm greenhouse to make them flower. According to fertility, 200 single plants were selected from the BC5 lines corresponding to some sterile S5F lines for self-pollination to obtain SBC5.
2. The rapid conversion method for radish sclerotium-cytoplasmic interaction male sterility lines as described in claim 1, characterized in that: The self-pollination and male-sterile line pair testcross process includes the following steps: (21) In the spring of the seventh year, 200 SBC5 plants were self-crossed to obtain S1, and at the same time, S1A was obtained by testcrossing with sterile plants in S5F. In the autumn, S1 and S1A were multiplied, and their marketability, disease resistance and quality were identified by sowing. Based on the autumn sowing identification results and fertility results, some sterile S1A lines and corresponding S1 lines that are similar to the materials to be converted were selected. 100 single plants of S1 line were self-crossed to obtain S2, and at the same time, S2A was obtained by testcrossing with the corresponding S1A. (22) In the eighth year, step (15) was repeated but autumn sowing identification was not performed, resulting in 100 self-pollinated materials S4 and the corresponding S4A; (23) In the spring of the ninth year, self-pollination and test crosses of materials continued. In the spring of this year, 100 individual plants of the all-sterile S5A line and the corresponding S5 were selected for self-pollination and test crosses for seed saving. The seed saving quantity of individual plants for self-pollination and test crosses was 150-200 seeds for autumn sowing and identification. In autumn, S5 and S5A were multiplied, and S5 and the corresponding test cross S5A were planted in open field. Based on commerciality, disease resistance and variety, S5 and S5A with similar recurrent parents were selected for self-pollination and test crosses to obtain S6 and S6A. S6 is the maintainer line and S6A is the sterile line. (24) In the spring of the tenth year, S6 and S6A were subjected to low temperature treatment, sown in the same net and mixed for seed retention, resulting in mixed S6 and S6A. They were then identified in autumn sowing. Thus, the male sterile line of Xinlimei material 173-4 was successfully converted.
3. A rapid conversion method for radish sclerotium-cytoplasmic interaction male sterility lines as described in claim 1 or 2, characterized in that: The aforementioned recurrent parent 173-4 is a stable inbred line bred through 8 generations of self-pollination and selection of the Xinlimei radish.