CN110800608A - Molecular breeding method of reproductive heterozygous female sterile restorer line and application thereof - Google Patents
Molecular breeding method of reproductive heterozygous female sterile restorer line and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic crop molecular biology, and particularly relates to a molecular breeding method of a reproductive heterozygous female sterile restorer line and application thereof. The invention relates to a method for breeding japonica rice carrying S5i or indica rice carrying S5j near-isogenic recovery strains by bridging a wide-affinity gene S5 n; introducing the temperature-sensitive sterile gene or the light-sensitive sterile gene into an S5i or S5j locus near isogenic restoring line to breed a near isogenic restoring line carrying the S5i + temperature-sensitive sterile gene or the light-sensitive sterile gene or the S5j + temperature-sensitive sterile gene or the light-sensitive sterile gene; the hybrid female sterile hybrid rice restorer line is bred in a seed production mode under the condition of high temperature or short sunlight. Because the restorer line bred by the invention is female sterile but male fertile, the male parent does not need to be removed after the pollination of the hybrid seeds, the mechanized harvesting of the hybrid seeds is realized, the seed production efficiency of the hybrid rice is improved, and the technical guarantee is provided for the mechanized seed production of the hybrid rice.
Description
Technical Field
The invention belongs to the technical field of genetic crop molecular biology, and particularly relates to a molecular breeding method of a reproductive heterozygous female sterile restorer line and application thereof.
Background
The invention, popularization and application of the hybrid rice make great contribution to the food safety of China and even the world. But the hybrid rice seed production has multiple links, complicated procedures, dependence on intensive labor force, high cost and low efficiency. With the continuous expansion of the urbanization construction scale in China, the rural labor population is sharply reduced. Therefore, the research and establishment of the hybrid rice seed production technology of the whole-course mechanized operation are particularly urgent.
At present, in the hybrid rice seed production process, the removal of the restorer line after pollination is an important factor influencing the whole mechanized operation of seed production. In the existing reports and patent applications, the technical idea about removing the male parent for seed production mainly focuses on the following aspects: firstly, separating hybrid seeds from male parents by utilizing the difference of the seed types of the male parents and the female parents through a mechanical sorting technology. The method only aims at the hybridization combination with larger particle type difference between the parent and the female parent, the particle type difference between the parent and the female parent of a plurality of hybridization combinations is smaller, particularly the combination with only particle length difference is difficult to effectively remove the mixed particle, and the technical difficulty of modifying parents by utilizing particle type specific genes (such as extra large particles and extra small particles) is large, so the method has large limitation. And secondly, separating the hybrid seeds from the male parents by a color selector by utilizing the difference of the glumes of the male parents and the female parents. A large number of practices show that the impurity removal capacity of the color sorter is limited, especially when the proportion of the impurity particles is large, the effect can be achieved only by repeatedly selecting the color for many times, the cost is high, the efficiency is difficult to improve, and therefore the application of the method is also limited. Thirdly, introducing herbicide sensitive genes into a restorer line, spraying herbicide to kill male parents after pollination in the flowering phase, and realizing mechanized mixed harvest (CN1318287, CN 102342243A). Practice shows that: the herbicide is sprayed only after pollination, all restoring lines are difficult to kill, even if the restoring lines are killed, a small amount of non-plump seeds with the germination capacity can be on the ears of the restoring lines, and in addition, the male parent and the female parent are mutually supported and held in a mixed planting state, so that the harvested hybrid seeds are inevitably doped with a small amount of male parent seeds. Fourthly, double elimination of restorer line seeds through the shell color and herbicide sensitivity characters, namely leading herbicide sensitive genes and shell color genes into the restorer line; or leading the herbicide sensitive gene into the male parent, leading the shell color gene into the female parent, and achieving the purpose of rejecting the male parent under the dual effects of herbicide spraying and color sorting. The method achieves ideal effect to a certain extent, but the spraying of the herbicide may cause influence on the ecological environment. In addition, the method has higher requirements on the sensitivity of the color selector. And fifthly, the female sterile line is used as a restorer line to carry out hybrid rice seed production (Gao Rong village and the like, 2012). However, the female sterile line reported at present is self-sexually propagated, so that the method is difficult to popularize and apply.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for breeding a reproductive heterozygous female sterile restorer line molecule and application thereof.
The invention is realized in this way, a breeding method of fertile heterozygous female sterile restorer line molecule, comprising: bridging with the wide-affinity gene S5n, and breeding japonica rice carrying S5i or indica rice carrying S5 j; introducing the temperature-sensitive sterile gene or the light-sensitive sterile gene into an S5i or S5j locus near isogenic restoring line to breed a near isogenic restoring line carrying the S5i + temperature-sensitive sterile gene or the light-sensitive sterile gene or the S5j + temperature-sensitive sterile gene or the light-sensitive sterile gene; the hybrid female sterile hybrid rice restorer line is bred in a seed production mode under the condition of high temperature or short sunlight.
Further, the temperature-sensitive sterile gene is tms5 gene, and the light-resistant genic male sterile gene is csa gene.
Furthermore, in the breeding and breeding process, a polymer for tracking a target gene is selected by using a molecular marker for assisting in selection, wherein molecular marker primers aiming at the tms5 gene, the csa gene and the S5 are shown in SEQ ID NO.1-SEQ ID NO. 6.
Further, the rice resource carrying the S5n gene is any of 02428, recurrent 422, and peiper 64.
Further, the rice resource carrying the S5i gene is any one of Huazhan, Yueya silk sprout, Minghui 63, Minghui 86, 9311, Shuhui 527, 9308, Mianax 725, Teqing, R288, Zhehui 7954.
Further, the rice resource carrying the S5j gene is any one of C57, C418, R18, wan hui No. 9, C fort, jin hui 58, R190, C4115, ning jing No.1, and wu jing No. 14.
Further, a temperature-sensitive sterile line carrying a temperature-sensitive sterile gene tms 5is Y58S or a homotypic line and a derivative temperature-sensitive genic sterile line thereof; the light sensitive sterile line carrying the light sensitive sterile gene csa is csa or its homotypic line and derivative light sensitive sterile line.
The molecular breeding method of the reproductive heterozygous female sterile restorer line can be applied to rice breeding.
Further, the bred fertile heterozygous female sterile restoring line is hybridized and matched with the three lines carrying the wide-affinity gene S5n or the two lines of sterile lines not carrying the temperature-sensitive sterile gene or the light-sensitive nuclear sterile gene to breed a new group of hybrid rice.
In summary, the advantages and positive effects of the invention are:
the invention is an innovation on the method, organically combines the molecular marking technology of modern molecular biology with a plurality of selection technologies such as hybridization, backcross, test cross and the like in the traditional crop genetic breeding, and provides a quick, accurate and foreseeable practical method for breeding new varieties of crops and creating new materials.
The invention uses wide-compatible gene S5n to bridge, uses indica-japonica hybrid sterile gene S5 and photo-thermo-sensitive genic sterile gene tms5 or csa to breed hybrid rice restorer line capable of reproducing heterozygous female sterility, namely uses wide-compatible gene S5n to bridge, replaces hybrid sterile gene S5 in the restorer line with indica-type equipotential S5i (or S5j) to japonica-type equipotential S5j (or S5i), and breeds near isogenic restorer line carrying S5j (or S5i) site; introducing a temperature-sensitive sterile gene tms5 (or a light-sensitive nuclear sterile gene csa) into an S5i or S5j site near-isogenic restoring line, namely breeding the near-isogenic restoring line carrying S5i + tms5 (or csa) or S5j + tms5 (or csa); the hybrid female sterile hybrid rice restorer line is bred in a seed production mode under the condition of high temperature or short sunlight. The obtained hybrid female sterile restoring line (S5iS5j + TMS5TMS5 or S5iS5j + CSACsa) iS hybridized and matched with a three-line sterile line carrying wide-affinity gene S5n or a two-line sterile line not carrying TMS5 or csa to breed a new hybrid rice combination. Because the bred restorer line is female sterile but male fertile, the male parent does not need to be removed after seed production and pollination, and the mechanized harvesting of seed production is realized, so that the seed production efficiency of hybrid rice is improved, and the technical guarantee is provided for the mechanized seed production of the hybrid rice.
The invention selects temperature sensitive sterile line to respectively carry out polymerization hybridization with photosensitive sterile line and material containing wide-affinity gene, and carries out molecular marker assisted selection and test matching with indica-japonica restoring line on the segregating generation. The rice thermo-sensitive genic male sterile line related by the invention comprises but is not limited to Y58S or the homologous line and the derivative thermo-sensitive genic male sterile line thereof. The rice light-reflecting sensitive genic male sterile line includes, but is not limited to csa or its homotypic line and derivative photosensitive genic male sterile line. The rice wide-affinity material includes, but is not limited to 02428, recurrent 422, peiper 64, etc. or a material carrying wide-affinity gene S5 n. The rice indica restorer line includes but is not limited to Huazhan, Yueyao si miao, Minghui 63, Minghui 86, 9311, Shuhui 527, 9308, Michelia angustifolia 725, Teqing, R288, Zhejiang hui 7954 and other materials. The rice japonica type restorer line comprises materials such as but not limited to C57, C418, R18, Anhui 9, C fort, Jinhui 58, R190, C4115, Ningjing No.1, Wuyujing No. 14 and the like.
Drawings
FIG. 1 is a molecular breeding technical route diagram of a hybrid female sterile restorer line;
FIG. 2 shows the amplification result of S5n linked marker on the selected partial S5n near isogenic restorer line;
FIG. 3 shows the results of partial strain amplification analysis of csa-linked molecular markers on the bred polymerizes S5i and csa;
FIG. 4 shows the amplification analysis result of tms5 linked molecular markers on selected partial strains of the polymeric S5i and tms 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Examples
The sterility of indica-japonica hybrid is an important form of rice reproductive isolation. At present, many loci of indica-japonica hybrid sterility are reported, and S5is one of the major effective loci. The site has 3 functional allelic forms, S5i is indica rice allelic form, S5j is japonica rice allelic form, and S5n is intermediate form, i.e., wide-affinity allelic form. The crossing of S5i and S5j allelic forms F1 female sterility (embryo sac sterility), while pollen development is normal, and S5i and S5j allelic forms and S5n can both fruit normally. According to the functional characteristics of different allelic types of the S5 locus, the cloned temperature-sensitive genic male sterile gene tms5(Zhou et al, 2012) or the light-sensitive genic male sterile gene csa (Zhang et al, 2013) is utilized, a reproducible heterozygous female sterile restorer line is created through hybrid backcross and a molecular marker selection means, the material basis and the technical support are improved for cultivating new hybrid rice varieties suitable for mechanical seed production, and the technical route is shown in figure 1.
The specific experimental process is as follows:
1. breeding of S5n near isogene carrying restoring strain
As the comprehensive agronomic characters of indica rice (carrying S5i) such as Huazhan and Yuehe shredded rice, or japonica rice restorer lines such as C418 (carrying S5j) are outstanding, and the application and popularization range in agricultural production is wider, Yuehe shredded rice is selected as recurrent parent, and is respectively hybridized and backcrossed with materials carrying wide affinity gene S5n such as 02428, and molecular marker screening (PC) is combinedR reaction system: 50ng of DNA; 5ul TAKARA rTaq buffer; 2ul of each upstream and downstream primer with the concentration of 10 mM; dNTPs 5ul at a concentration of 2.5 mM; TAKARA rTaq 5U; add ddH2The amount of O is 50 ul. PCR procedure: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 55 ℃, 30sec at 72 ℃, 32 cycles; 72 ℃ for 5min), breeding the Guangdong grass silk seedling line homozygous at the site of the broad-affinity gene S5n in the segregation population backcrossed for 4-5 generations.
2. Breeding of indica near isogenic polymeric strain carrying tms5 (or csa) + S5i
Taking YueHe Silvery (carrying S5i) as recurrent parent, hybridizing with Y58S, csa, etc., backcrossing, and combining molecular marker screening (PCR reaction system: 50ng DNA; 5ul TAKARA rTaq buffer; 2ul each of 10mM upstream and downstream primers; 5ul of 2.5mM dNTPs; TAKARA rTaq 5U; ddH2The amount of O is 50 ul. PCR procedure: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 56 ℃, 30sec at 72 ℃ and 32 cycles; 72 ℃ for 5min), breeding a polymeric strain carrying tms5 (or csa) + S5i in a segregation population of backcross 4-5 generations.
3. Breeding of indica rice near-isogenic recovery line carrying S5j
The Yuehe grass silkworms carrying the S5n gene are recurrent parents, hybridized with C418 carrying S5j resource, backcrossed and combined with molecular marker screening (PCR reaction system: 50ng DNA; 5ul TAKARA rTaq buffer; 2ul each of 10mM upstream and downstream primers; 5ul of 2.5mM dNTPs; TAKARA rTaq 5U; ddH added2The amount of O is 50 ul. PCR procedure: 3min at 95 ℃; 30sec at 95 ℃, 30sec at 56 ℃, 30sec at 72 ℃ and 32 cycles; 72 ℃ for 5min), and breeding the Yuexi silkworms carrying S5j in the segregation population of backcross 4-5 generations.
4. Breeding of fertile heterozygous female sterile restoring line
Under the condition of high temperature or short sunshine, the Yue He Si Miao TMS5TMS 5iS taken as a female parent and the restorer line Yue He Si Miao S5jS5j iS hybridized to produce seeds, and the Yue He Si Miao S5iS5j + TMS5TMS 5iS propagated. And planting Yuexi silkworms S5iS5j + TMS5TMS5 in the compost and Hainan Ling water respectively, and inspecting pollen fertility and seed setting rate, wherein the results are shown in a table 1, which shows that the Yuexi silkworms S5iS5j + TMS5TMS5 are female sterile.
TABLE 1 heterozygosis female restorer line in YueSemian with Lingshui pollen stainability and seed set rate
5. Seed production application of fertile heterozygous female sterile restoring line
Two-line sterile line carrying S5n iS used for cultivating dwarf 64S and Yueyao si miao S5iS5j + TMS5TMS5 hybrid seed production, the male parent and the female parent are mechanically harvested together after the hybrid seed iS mature, and the purity planting identification iS carried out on the hybrid seeds harvested together. And performing field purity identification on 500 dwarf plants 64S/(Guangdong grass silk seedlings S5iS5j + TMS5TMS5) in 5 months in 2019 after 3 times of combined fertilization planting in 5 months, wherein the results are shown in a table 2, the purities are all more than 96% (purity reference line of hybrid rice), and the results show that the heterozygous female sterile line Guangdong grass silk seedlings S5iS5j + TMS5TMS5 can be used as a restoring line for mechanical seed production of hybrid rice.
Table 2 results of field identification of hybrid combination culture of 64S/(S5 iS5j + TMS5TMS5) purity
The molecular marker assisted selection of the invention is to use the genome DNA of the single plant with excellent separation generation as a template and carry out PCR analysis by using double primers with certain nucleotide sequences (5 '-3') so as to trace and select the aggregate of target genes. The primer sequences are as follows:
the map of partial molecular marker breeding is shown in fig. 2-4, wherein fig. 2 is the amplification result of S5n linked marker on the bred partial S5n near isogenic restorer line; m is DNA Marker; 02428; 2, Yueye shredded seedlings; 02428 part of Yueya silkworms F1Carrying out single plant cultivation; s5n near isogenic restorer lines 4-11; FIG. 3 is a partial strain amplification analysis node of csa-linked molecular marker pair of the bred polymeric S5i and csaFruit; m is DNA Marker; CSA: CSA short-day sterile line; y is YueSemian; csa A/Yue He Si Miao F1Carrying out single plant cultivation; 1-5: csa and S5i polymeric lines; FIG. 4 shows the amplification analysis result of tms5 linked molecular markers on selected partial strains of the polymeric S5i and tms 5; m is DNA Marker; 1-7S 5i and tms5 polymeric lines; 8, Yueye shredded seedlings; 9: Y58S/Yue He Si Miao F1Carrying out single plant cultivation; Y58S.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> institute of Paddy Rice of agricultural science institute of Anhui province
A limited liability company of Huaan species of Anhui province
<120> molecular breeding method of fertile heterozygous female sterile restorer line and application thereof
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<213> Artificial sequence (csa-F)
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<213> Artificial sequence (tms5-R)
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Claims (8)
1. A method for breeding a reproductive heterozygous female sterile restorer line molecule is characterized by comprising the following steps: bridging with the wide-affinity gene S5n, and breeding japonica rice carrying S5i or indica rice carrying S5 j; introducing the temperature-sensitive sterile gene or the light-sensitive sterile gene into an S5i or S5j locus near isogenic restoring line to breed a near isogenic restoring line carrying the S5i + temperature-sensitive sterile gene or the light-sensitive sterile gene or the S5j + temperature-sensitive sterile gene or the light-sensitive sterile gene; the hybrid female sterile hybrid rice restorer line is bred in a seed production mode under the condition of high temperature or short sunlight.
2. The molecular breeding method of a fertile heterozygous female sterile restorer line according to claim 1, characterized in that: the temperature-sensitive sterile gene is tms5 gene, and the light-resistant genic male sterile gene is csa gene.
3. The molecular breeding method of a fertile heterozygous female sterile restorer line according to claim 2, characterized in that: in the breeding and breeding process, a polymer for tracking a target gene is selected by using a molecular marker for assisting selection, wherein molecular marker primers aiming at the tms5 gene, the csa gene and the S5 are shown in SEQ ID NO.1-SEQ ID NO. 6.
4. The molecular breeding method of a fertile heterozygous female sterile restorer line according to claim 1, characterized in that: the rice resource carrying the S5n gene is any one of 02428, recurrent 422 and Pan 64.
5. The molecular breeding method of a fertile heterozygous female sterile restorer line according to claim 1, characterized in that: the rice resource carrying S5i gene is any one of Huazhan, Yueye miao, Minghui 63, Minghui 86, 9311, Shuhui 527, 9308, Mianan 725, Teqing, R288, Zhejiang hui 7954.
6. The molecular breeding method of a fertile heterozygous female sterile restorer line according to claim 1, characterized in that: the rice resource carrying the S5j gene is any one of C57, C418, R18, Anhui Hui No. 9, C fort, Jinhui 58, R190, C4115, Ningjing No.1 and Wuyujing No. 14.
7. The molecular breeding method of a fertile heterozygous female sterile restorer line according to claim 2, characterized in that: the temperature-sensitive sterile line carrying the temperature-sensitive sterile gene tms 5is Y58S or the homologous line and the derivative temperature-sensitive genic sterile line thereof; the light sensitive sterile line carrying the light sensitive sterile gene csa is csa or its homotypic line and derivative light sensitive sterile line.
8. Use of a method of molecular breeding a fertile heterozygous female sterile restorer line according to any one of claims 1 to 7 in rice breeding.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112243855A (en) * | 2020-10-22 | 2021-01-22 | 重庆市农业科学院 | Breeding method of high-quality hybrid rice suitable for mechanical seed production |
| CN112772404A (en) * | 2020-12-31 | 2021-05-11 | 云南农业大学 | Breeding method of rice female nuclear sterility restoring line |
| CN113728911A (en) * | 2021-03-12 | 2021-12-03 | 华南农业大学 | Novel rice high-temperature reversible sterile line and two-line hybrid rice seed production system |
| CN115943885A (en) * | 2022-12-20 | 2023-04-11 | 安徽省农业科学院水稻研究所 | Molecular breeding method for converting rice positive-temperature-sensitive sterile line into fertile heterozygous male sterile line and application of molecular breeding method |
| CN116019010A (en) * | 2023-01-30 | 2023-04-28 | 安徽省农业科学院水稻研究所 | Molecular breeding method for converting rice inverse thermosensitive sterile line into reproducible heterozygous male sterile line and application thereof |
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| CN102599044A (en) * | 2012-03-14 | 2012-07-25 | 安徽省农业科学院水稻研究所 | Breeding method of wide-compatibility photo-thermo sensitive sterile rice pyramiding lines |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102599044A (en) * | 2012-03-14 | 2012-07-25 | 安徽省农业科学院水稻研究所 | Breeding method of wide-compatibility photo-thermo sensitive sterile rice pyramiding lines |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112243855A (en) * | 2020-10-22 | 2021-01-22 | 重庆市农业科学院 | Breeding method of high-quality hybrid rice suitable for mechanical seed production |
| CN112243855B (en) * | 2020-10-22 | 2022-03-11 | 重庆市农业科学院 | Breeding method of high-quality hybrid rice suitable for mechanical seed production |
| CN112772404A (en) * | 2020-12-31 | 2021-05-11 | 云南农业大学 | Breeding method of rice female nuclear sterility restoring line |
| CN112772404B (en) * | 2020-12-31 | 2021-07-27 | 云南农业大学 | Breeding method of rice female nuclear sterility restoring line |
| CN113728911A (en) * | 2021-03-12 | 2021-12-03 | 华南农业大学 | Novel rice high-temperature reversible sterile line and two-line hybrid rice seed production system |
| CN113728911B (en) * | 2021-03-12 | 2022-05-31 | 华南农业大学 | Novel rice high-temperature reversible sterile line and two-line hybrid rice seed production system |
| CN115943885A (en) * | 2022-12-20 | 2023-04-11 | 安徽省农业科学院水稻研究所 | Molecular breeding method for converting rice positive-temperature-sensitive sterile line into fertile heterozygous male sterile line and application of molecular breeding method |
| CN116019010A (en) * | 2023-01-30 | 2023-04-28 | 安徽省农业科学院水稻研究所 | Molecular breeding method for converting rice inverse thermosensitive sterile line into reproducible heterozygous male sterile line and application thereof |
| CN116019010B (en) * | 2023-01-30 | 2023-11-24 | 安徽省农业科学院水稻研究所 | Molecular breeding method for converting rice inverse thermosensitive sterile line into reproducible heterozygous male sterile line and application thereof |
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