CN110800608A - Molecular breeding method of reproductive heterozygous female sterile restorer line and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology of genetic crops, and in particular relates to a molecular breeding method for a fertile heterozygous female sterile restorer line and its application. In the present invention, the broad affinity gene S5n is used as a bridge to select and breed japonica rice carrying S5i or indica rice carrying S5j near-equivalent restorer lines; the temperature-sensitive sterility gene or the photosensitive nuclear sterility gene is introduced into the S5i or S5j site near-equivalent Among the gene restorer lines, a near-isogenic restorer line carrying the S5i+ temperature-sensitive sterility gene or the reverse light-sensitive sterility gene, or the S5j+ temperature-sensitive sterility gene or the reverse-light-sensitive sterility gene is bred; seed production under high temperature or short-day conditions , to realize the reproduction of heterozygous female sterile hybrid rice restorer lines. Since the females of the restorer line selected by the invention are sterile but the males are fertile, the male parent does not need to be removed after seed production and pollination, thus realizing the mechanized harvesting of seed production, improving the efficiency of hybrid rice seed production, and providing technical support for the mechanized hybrid rice seed production.

Description

A molecular breeding method for a reproductive heterozygous female sterile restorer line and its application

technical field

The invention belongs to the technical field of molecular biology of genetic crops, and in particular relates to a molecular breeding method for a fertile heterozygous female sterile restorer line and its application.

Background technique

The invention, popularization and application of hybrid rice have made significant contributions to food security in my country and even in the world. However, the production of hybrid rice seeds has many links and complicated procedures, and it relies on intensive labor, which has high cost and low efficiency. With the continuous expansion of the scale of urbanization in my country, the rural labor force has dropped sharply. Therefore, the research and establishment of hybrid rice seed production technology with full mechanized operation is particularly urgent.

At present, in the process of hybrid rice seed production, the removal of restorer lines after pollination is an important factor affecting the mechanized operation of the whole seed production process. In the existing reports and patent applications, the technical concept of removing the male parent for seed production mainly focuses on the following aspects: 1. Using the difference in the grain size of the parent parent to realize the separation of the hybrid from the male parent through mechanical sorting technology. This method can only be used for the hybrid combinations with large differences in the parental grain type, and many hybrid combinations have small parental grain type differences, especially for the combinations with only grain length differences, it is difficult to effectively eliminate the hybrid, and the use of grain type-specific genes ( Such as extra-large grains, extra-small grains), the technical difficulty of transforming parents is great, so this method has great limitations. 2. Use the color difference of the parental glume to separate the hybrid from the male parent through a color sorter. A large number of practices have shown that the color sorter has a limited ability to remove impurities, especially when the proportion of impurities is large, it often requires repeated color sorting to be effective, the cost is high, and the efficiency is difficult to improve, so the application of this method is also limited. 3. The herbicide-sensitive gene is introduced into the restorer line, and the herbicide is sprayed to kill the male parent after pollination at the flowering stage to realize mechanized mixed harvesting (CN1318287, CN102342243A). Practice has shown that it is often difficult to kill all restorer lines by spraying herbicides after pollination alone. Even if the restorer lines are killed, there may be a small amount of non-full seeds with germination ability on the ears, and the parents are mixed. Under the mutual support and support, there is inevitably a small amount of male parent seeds mixed in the harvested hybrids. 4. Double-knock out restorer seeds by shell color and herbicide sensitivity traits, that is, introduce herbicide sensitive gene and shell color gene into restorer line; or introduce herbicide sensitive gene into male parent, and transfer shell color gene into female parent, Under the dual action of herbicide spraying and color selection, the purpose of eliminating male parents is achieved. This method achieves ideal results to a certain extent, but the spraying of herbicides may have an impact on the ecological environment. In addition, this method has higher requirements on the sensitivity of the color sorter. 5. Use female sterile lines as restorer lines for hybrid rice seed production (Gao Rongcun et al., 2012). However, the female sterile lines reported so far are all self-reproductive, so this method is difficult to popularize and apply.

SUMMARY OF THE INVENTION

Aiming at the problems existing in the prior art, the present invention provides a molecular breeding method for a fertile heterozygous female sterile restorer line and its application.

The present invention is achieved in this way, a molecular breeding method for reproductive heterozygous female sterile restorer lines, comprising: bridging with broad affinity gene S5n, selecting and breeding japonica carrying S5i or indica near-equivalent restorer lines carrying S5j ;Introduce the thermosensitive sterility gene or the photosensitive nuclear sterility gene into the near-isogenic restorer line at the S5i or S5j locus, and bred to carry the S5i+ thermosensitive sterility gene or the photosensitive sterility gene, or the S5j+ thermosensitive sterility gene Or near-isogenic restorer lines of reflective photosensitive sterile genes; the reproduction of heterozygous female sterile hybrid rice restorer lines can be achieved by seed production under high temperature or short-day conditions.

Further, the temperature-sensitive sterility gene is the tms5 gene, and the light-reflecting nuclear sterility gene is the csa gene.

Further, in the breeding and selection process, molecular marker-assisted selection is used to track the aggregate of the target gene, wherein the molecular marker primers for tms5 gene, csa gene and S5 are shown in SEQ ID NO.1-SEQ ID NO.6.

Further, the rice resource carrying the S5n gene is any one of 02428, Samsara 422, and Pei'ai 64.

Further, the rice resource carrying the S5i gene is any one of Huazhan, Yuehe Simiao, Minghui 63, Minghui 86, 9311, Shuhui 527, 9308, Mianhui 725, Teqing, R288, and Zhehui 7954 .

Further, the rice resource carrying the S5j gene is any one of C57, C418, R18, Wanhui 9, Cbao, Jinhui 58, R190, C4115, Ningjing 1, and Wuyunjing 14.

Further, the temperature-sensitive sterile line carrying the temperature-sensitive sterile gene tms5 is Y58S or its isotype and the derived temperature-sensitive sterile line; the light-sensitive sterile line carrying the anti-photosensitive sterile gene csa gene is csa or its isotype line and derived photosensitive sterile line.

Application of the above-mentioned molecular breeding method of a reproductive heterozygous female sterile restorer line in rice breeding.

Further, the selected fertile heterozygous female sterile restorer line is crossed and matched with a three-line carrying the broad-compatibility gene S5n, or with a two-line sterile line that does not carry a temperature-sensitive sterile gene or a photosensitive nuclear sterile gene. , Breeding a new group of hybrid rice.

To sum up, the advantages and positive effects of the present invention are:

The invention is a method innovation, which organically combines the molecular marker technology of modern molecular biology and the traditional crop genetic breeding such as hybridization, backcrossing, test crossing and other selection technologies, so as to provide new crop varieties breeding, The creation of new materials provides a fast, accurate and predictable practical method.

In the present invention, the broad-compatibility gene S5n is used as a bridge, and the indica-japonica hybrid sterility gene S5 and the photothermo-sensitive nuclear sterility gene tms5 or csa are used to cultivate a reproducible heterozygous female sterile hybrid rice restorer line. S5n bridges the hybrid sterility gene S5 in the restorer line by replacing the indica allele S5i (or S5j) with the japonica allele S5j (or S5i), and bred to carry the S5j (or S5i) site. Gene restorer line; the temperature-sensitive sterility gene tms5 (or the photosensitive nuclear sterility gene csa) was introduced into the near-isogenic restorer line at the S5i or S5j locus, that is, it was bred to carry S5i+tms5 (or csa) or S5j+tms5 ( or csa) near-isogenic restorer lines; the reproduction of heterozygous female sterile hybrid rice restorer lines is achieved by seed production under high temperature or short-day conditions. The obtained heterozygous female sterile restorer line (S5iS5j+TMS5tms5 or S5iS5j+CSAcsa) is then crossed with a three-line carrying the broad compatibility gene S5n or a two-line male sterile line not carrying tms5 or csa to breed a new hybrid rice combination. . Since the females of the selected restorer lines are sterile but the males are fertile, there is no need to remove the male parent after seed production and pollination, and the mechanized harvesting of seed production is realized, thus improving the efficiency of hybrid rice seed production and providing technical support for the mechanized seed production of hybrid rice.

In the present invention, the temperature-sensitive sterile line is selected to carry out the polymerization hybridization with the photosensitive sterile line and the material containing the broad-compatibility gene respectively, and the separated generations are subjected to molecular marker-assisted selection and matching with the indica-japonica restorer line. The rice thermogenic sterile line involved in the present invention includes, but is not limited to, Y58S or its isotype line and derived thermogenic sterile line. The reflective GMS lines of rice include but are not limited to csa or its isotypes and derived GMS lines. Rice broad-compatibility materials include, but are not limited to, 02428, Samsara 422, Pei'ai 64, etc. or materials carrying the broad-compatibility gene S5n. Rice indica restorer lines include but are not limited to Huazhan, Yuehesimiao, Minghui 63, Minghui 86, 9311, Shuhui 527, 9308, Mianhui 725, Teqing, R288, Zhehui 7954 and other materials. Rice japonica restorer lines include but are not limited to C57, C418, R18, Wanhui No. 9, Cbao, Jinhui 58, R190, C4115, Ningjing No. 1, Wuyunjing No. 14 and other materials.

Description of drawings

Fig. 1 is the technical roadmap of molecular breeding of heterozygous female sterile restorer lines;

Fig. 2 is the amplification result of S5n-linked marker to selected partial S5n near-isogenic restorer lines;

Fig. 3 is the amplification analysis result of csa-linked molecular markers on selected strains of aggregated S5i and csa;

Fig. 4 is the result of amplification analysis of selected strains of aggregated S5i and tms5 by tms5-linked molecular markers.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels unless otherwise specified. The specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention.

Example

Indica-japonica hybrid sterility is an important form of reproductive isolation in rice. At present, many indica-japonica hybrid sterility loci have been reported, and S5 is one of the major loci. There are three functional alleles of this locus, S5i is an indica allele, S5j is a japonica allele, and S5n is an intermediate or broad-affinity allele. S5i allele and S5j allele crossed F1 female sterile (embryonic sac sterile), but normal pollen development, S5i and S5j alleles and S5n, can be normal fruit. The present invention is based on the functional properties of different alleles of the above-mentioned S5 site, and utilizes the cloned thermosensitive sterility gene tms5 (Zhou et al., 2012) or the photosensitive sterility gene csa (Zhang et al., 2013), through hybridization Backcrossing and molecular marker selection methods are used to create a reproducible heterozygous female sterile restorer line, which improves the material basis and technical support for cultivating new hybrid rice varieties suitable for mechanized seed production. The technical route is shown in Figure 1.

The specific experimental process is as follows:

1. Breeding of near-isogenic restorer lines carrying S5n

In view of the outstanding comprehensive agronomic characters of indica rice (carrying S5i) or C418 (carrying S5j) and other japonica restorer lines such as Huazhan and Yuehesimiao, and the wide range of application and promotion in agricultural production, Yuehesimiao was selected as the The recurrent parents were hybridized and backcrossed with the material carrying the broad-affinity gene S5n, such as 02428, and combined with molecular marker screening (PCR reaction system: 50ng DNA; 5ul TAKARA rTaq buffer; the concentration of 10mM upstream and downstream primers was 2ul each; the concentration was 2.5 mM dNTPs 5ul; TAKARA rTaq 5U; add ddH ₂ O to make up to 50ul. PCR program: 95°C 3min; 95°C 30sec, 55°C 30sec, 72°C 30sec, 32 cycles; 72°C 5min), backcross 4～ The 5 generations of segregating populations were selected and bred the Yuehe silk seedling line homozygous for the S5n locus of the broad affinity gene.

2. Breeding of indica near-isogenic aggregation lines carrying tms5 (or csa) + S5i

Taking Yuehe silk seedlings (carrying S5i) as the recurrent parent, hybridized with Y58S, csa, etc., respectively, backcrossed, combined with molecular marker screening (PCR reaction system: 50ng DNA; 5ul TAKARA rTaq buffer; the concentration is 10mM upstream and downstream primers each 2ul; 5ul of dNTPs with a concentration of 2.5mM; 5U of TAKARA rTaq; add ddH ₂ O to make up to 50ul. PCR program: 95°C 3min; 95°C 30sec, 56°C 30sec, 72°C 30sec, 32 cycles; 72°C 5min), in the back The aggregated strains carrying tms5 (or csa)+S5i were selected from the segregated population of 4-5 generations.

3. Breeding of near-equivalent restorer lines of indica rice carrying S5j

The Yuehe silk seedlings that have carried the S5n gene are the recurrent parents, hybridized with the S5j resource C418, backcrossed, and combined with molecular marker screening (PCR reaction system: 50ng DNA; 5ul TAKARA rTaq buffer; the concentration is 10mM upstream and downstream primers each 2ul; 5ul of dNTPs with a concentration of 2.5mM; 5U of TAKARA rTaq; add ddH ₂ O to make up to 50ul. PCR program: 95°C for 3min; 95°C for 30sec, 56°C for 30sec, 72°C for 30sec, 32 cycles; 72°C for 5min). The Yuehe silk seedlings carrying S5j were selected from the segregated population of 4-5 generations.

4. Breeding of fertile heterozygous female sterile restorer lines

Under the condition of high temperature or short-day light, the Yuehe silk seedling tms5tms5 was used as the female parent, and it was crossed with the restorer line Yuehe silk seedling S5jS5j, and the Yuehe silk seedling S5iS5j+TMS5tms5 was propagated. Yuehe silk seedlings S5iS5j+TMS5tms5 were planted in Hefei and Hainan Lingshui respectively, and pollen fertility and seed setting rate were investigated.

Table 1 The pollen dyeability and seed setting rate of the heterozygous female restorer lines of Yuehe silk seedlings in Hefei and Lingshui

5. Seed Production Application of Reproducible Heterozygous Female Sterile Restorer Line

The two sterile lines carrying S5n, Pei'ai 64S, and Yuehe Simiao S5iS5j+TMS5tms5 were used for hybrid seed production. In May 2019, the hybrid combination Pei'ai 64S/(Yuehe Simiao S5iS5j+TMS5tms5) was planted in Hefei, 500 plants, 3 replicates, and the field purity identification was carried out in August 2019. The results are shown in Table 2, and the purity is >96% (hybrid rice Purity baseline), indicating that the heterozygous female sterile line Yuehe Simiao S5iS5j+TMS5tms5 can be used as a restorer line for mechanized seed production of hybrid rice.

Table 2 The results of field identification of the purity of the hybrid combination Pei'ai 64S/(Yuehe Simiao S5iS5j+TMS5tms5)

The molecular marker-assisted selection in the present invention takes the genomic DNA of the isolated generation of excellent individual plants as a template, and uses double primers with a certain nucleotide sequence (5'-3') to carry out PCR analysis to track and select the aggregate of the target gene. The primer sequences are as follows:

The map of partial molecular marker selection is shown in Fig. 2-Fig. 4, wherein Fig. 2 is the amplification result of S5n-linked marker to selected partial S5n near-isogenic restorer lines; M:DNA Marker;1:02428;2:Guangdong Hesi Miao; 3: 02428/Yue He Si Miao F ₁ individual plant; 4-11: S5n near-isogenic restorer line; Fig. 3 is the partial line amplification of the selected aggregated S5i and csa by csa-linked molecular markers Analysis results; M: DNA Marker; CSA: csa short-day sterile line; Y: Yuehe silk seedling; H: csa/Yuehe silk seedling F ₁ single plant; 1-5: csa and S5i aggregated line; Figure 4 tms5-linked molecular marker is the result of the amplification analysis of the selected lines of aggregated S5i and tms5; M: DNA Marker; 1-7: S5i and tms5 aggregated lines; 8: Yuehe silk seedlings; 9: Y58S/Guangdong Hesi Miao F ₁ single plant; 10: Y58S.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Rice Research Institute, Anhui Academy of Agricultural Sciences

Anhui Huaan Seed Industry Co., Ltd.

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Claims (8)

1. a method for molecular breeding of a fertile heterozygous female sterile restorer line, is characterized in that, comprising: bridging with broad affinity gene S5n, selecting and breeding the japonica rice carrying S5i or the indica rice carrying S5j near and other restorer lines; The temperature-sensitive sterility gene or the photosensitive nuclear sterility gene was introduced into the near-isogenic restorer line at the S5i or S5j locus, and it was bred to carry the S5i+ temperature-sensitive sterility gene or the photo-sensitive sterility gene, or the S5j+ temperature-sensitive sterility gene or A near-isogenic restorer line with a reflective photosensitive sterile gene; the reproduction of a heterozygous female sterile hybrid rice restorer line can be achieved by seed production under high temperature or short-day conditions. 2. a kind of molecular breeding method that can breed heterozygous female sterile restorer line according to claim 1, it is characterized in that: described temperature-sensitive sterile gene is tms5 gene, and described light-reflecting nuclear sterile gene is csa Gene. 3. a kind of molecular breeding method of fertile heterozygous female sterile restorer line according to claim 2, is characterized in that: utilize molecular marker assisted selection to track the aggregate of target gene in the breeding and breeding process, wherein for tms5 The molecular marker primers of the gene, csa gene and S5 are shown in SEQ ID NO.1-SEQ ID NO.6. 4 . The molecular breeding method for a fertile heterozygous female sterile restorer line according to claim 1 , wherein the rice resource carrying the S5n gene is any one of 02428, Samsara 422, and Pei’ai 64. 5 . 5. a kind of molecular breeding method that can breed heterozygous female sterile restorer lines according to claim 1, is characterized in that: the rice resource carrying S5i gene is Huazhan, Yuehe silk seedling, Minghui 63, Minghui Any of 86, 9311, Shuhui 527, 9308, Mianhui 725, Teqing, R288, and Zhejiang 7954. 6. a kind of molecular breeding method that can breed heterozygous female sterile restorer line according to claim 1, is characterized in that: the rice resource that carries S5j gene is C57, C418, R18, Wanhui No. 9, C Fort, Any of Jinhui 58, R190, C4115, Ningjing No. 1 and Wuyunjing No. 14. 7. a kind of molecular breeding method of fertile heterozygous female sterile restorer line according to claim 2, is characterized in that: the thermosensitive sterile line carrying thermosensitive sterile gene tms5 is Y58S or its homotype line and derivative Thermosensitive sterile line; the photosensitive sterile line carrying the photosensitive gene csa gene is csa or its isotype and derivative photosensitive sterile line. 8. The application of the molecular breeding method of a reproductive heterozygous female sterile restorer line as described in any one of claims 1-7 in rice breeding.

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