Method for losing high-temperature rejuvenation phenotype of wild abortion type cytoplasmic male sterile line rice sensitive to high temperature

Abstract

The invention discloses a method for losing high-temperature rejuvenation phenotypes of wild abortion type cytoplasmic male sterile line rice sensitive to high temperature. The method comprises the following steps: replacing a DNA fragment Rf3-TOS in the wild abortion type cytoplasmic male sterile line rice sensitive to high temperature into a DNA fragment Rf3-nonTOS, so that the high-temperature rejuvenation phenotype of the wild abortion type cytoplasmic male sterile line rice sensitive to high temperature is lost; the nucleotide sequence of the DNA fragment Rf3-TOS is shown as SEQ ID No. 1 in a sequence table; the nucleotide sequence of the DNA fragment Rf3-nonTOS is as shown in SEQ ID No.2 in a sequence table; the wild abortion type cytoplasmic male sterile line rice sensitive to high temperature is wild abortion type cytoplasmic male sterile line rice with a high-temperature restoration phenotype. The invention lays a solid foundation for the safety of hybrid rice seed production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology breeding, and in particular to a method for making a wild-type cytoplasmic male sterile rice line that is sensitive to high temperature lose its high-temperature restoration phenotype. Background Art

[0002] The male sterile rice line is a special type of rice. In its own floral organs, the male organs are not fully developed and cannot form normal pollen, while its female organs develop normally. Therefore, it cannot reproduce on its own and needs the help of foreign rice pollen to produce seeds. There are many types of rice cytoplasmic male sterility, including wild abortion type, red lotus type, Baotai type, etc. Among them, the discovery of wild abortion type cytoplasmic male sterile (CMS-WA) wild rice has realized the matching and large-scale utilization of the "three lines" of hybrid rice for the first time, and is the most important type of sterile line used in the three-line hybrid rice.

[0003] In production practice, it was found that some wild-type cytoplasmic male sterile lines have the phenomenon of high-temperature re-fertilization. For example, Tianfeng A is the female parent of Tianyou Huazhan, a high-quality and high-yield indica three-line hybrid rice combination, and has the characteristics of high-temperature re-fertilization. The instability of its fertility has brought great risks to hybrid rice seed production. The seed production problem of Tianyou Huazhan has always been a bottleneck for its promotion and application. Over the years, seed production problems have continued to occur in production, and it is difficult to produce enough qualified hybrid rice seeds. Relevant studies have shown that under natural high temperature conditions, the pollen fertility of the sterile lines Zhenshan 97A, T98A, Fengyuan A and Gang 46A is stable; Long 398A, Longxiang 634A, 4302A, You IA and D62A will have a small amount of dyeable pollen under high temperature conditions; and Tianfeng A, Wufeng A and Yuetai A will also have obvious high-temperature re-fertilization under high temperature conditions, which is risky in production and application. Therefore, overcoming the high-temperature re-fertilization of wild-type cytoplasmic male sterile lines is of great significance to the safe production of hybrid rice seeds and ensuring food security. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to make the high-temperature restoration phenotype of wild-type cytoplasmic male sterile rice lines that are sensitive to high temperature lose.

[0005] In order to solve the above technical problems, the present invention first provides a method for causing the wild-type cytoplasmic male sterile rice line to lose its high-temperature restoration phenotype that is sensitive to high temperature.

[0006] The method provided by the present invention causes the high-temperature refertility phenotype of wild-type cytoplasmic male sterile rice sensitive to high temperature to be lost, comprising: replacing a DNA fragment Rf3-TOS in the wild-type cytoplasmic male sterile rice sensitive to high temperature with a DNA fragment Rf3-nonTOS, so that the high-temperature refertility phenotype of the wild-type cytoplasmic male sterile rice sensitive to high temperature is lost; the nucleotide sequence of the DNA fragment Rf3-TOS is SEQ ID No. 1 in the sequence list; the nucleotide sequence of the DNA fragment Rf3-nonTOS is SEQ ID No. 2 in the sequence list; the wild-type cytoplasmic male sterile rice sensitive to high temperature is a wild-type cytoplasmic male sterile rice with a high-temperature refertility phenotype.

[0007] In the above method, the DNA fragment Rf3-TOS in the wild abortion type cytoplasmic male sterile rice line that is sensitive to high temperature is replaced by the DNA fragment Rf3-nonTOS by hybridizing the improved maintainer line with the wild abortion type cytoplasmic male sterile rice line that is sensitive to high temperature, and then backcrossing is achieved; the improved maintainer line is obtained by replacing the DNA fragment Rf3-TOS in the maintainer line rice corresponding to the wild abortion type cytoplasmic male sterile rice line that is sensitive to high temperature with the DNA fragment Rf3-nonTOS.

[0008] In the above method, the method of replacing the DNA fragment Rf3-TOS in the maintainer rice corresponding to the wild-type cytoplasmic male sterile line that is sensitive to high temperature with the DNA fragment Rf3-nonTOS comprises the following steps:

[0009] 1) Using the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line sensitive to high temperature as the female parent and the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line insensitive to high temperature as the male parent to obtain F1 plants, and selfing the F1 plants to obtain selfed offspring; the wild abortion type cytoplasmic male sterile line insensitive to high temperature is a wild abortion type cytoplasmic male sterile line rice without high temperature re-fertilization phenotype; the wild abortion type cytoplasmic male sterile line sensitive to high temperature and the maintainer line corresponding to the wild abortion type cytoplasmic male sterile line sensitive to high temperature both contain the DNA fragment Rf3-TOS; the wild abortion type cytoplasmic male sterile line insensitive to high temperature and the maintainer line corresponding to the wild abortion type cytoplasmic male sterile line insensitive to high temperature both contain the DNA fragment Rf3-nonTOS fragment.

[0010] 2) Selecting a single plant carrying the DNA fragment Rf3-nonTOS but not carrying the DNA fragment Rf3-TOS from the selfed progeny obtained in step 1) as the female parent, and performing backcrossing with the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line that is sensitive to high temperature as the male parent, and then continuously selfing-backcrossing, and then continuously selfing, selecting a single plant carrying the DNA fragment Rf3-nonTOS but not carrying the DNA fragment Rf3-TOS from the selfed progeny to obtain an improved maintainer line, thereby completing the replacement of the DNA fragment Rf3-TOS in the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line that is sensitive to high temperature with the DNA fragment Rf3-nonTOS.

[0011] In the above method, in 2), the selfing-backcrossing is first selfing once and then backcrossing once.

[0012] In the above method, in 2), the number of consecutive self-pollination-backcrossing is at least 5 times, and the backcrossing mother in the self-pollination-backcrossing is a single plant whose plant and leaf morphology is similar to the maintenance line corresponding to the wild abortion type cytoplasmic male sterile line that is sensitive to high temperature and carries the DNA fragment Rf3-nonTOS but does not carry the DNA fragment Rf3-TOS.

[0013] In the above method, in 2), the number of consecutive selfing is at least 3 times, such as 3 times, 4 times or 5 times. The selfing parent is a single plant with plant and leaf morphology similar to the maintainer line corresponding to the wild abortion type cytoplasmic male sterile line sensitive to high temperature and carrying the DNA fragment Rf3-nonTOS but not carrying the DNA fragment Rf3-TOS.

[0014] In the above method, the plant and leaf morphology may include phenotypes such as plant height, leaf freshness, stem strength, ear fading and / or fruit setting rate.

[0015] In the above method, the wild-type cytoplasmic male sterile line that is sensitive to high temperature is Tianfeng A, Wufeng A, Jinlong A or Longjing 4302A.

[0016] In the above method, the maintenance line corresponding to the wild-type cytoplasmic male sterile line that is sensitive to high temperature is Tianfeng B, Wufeng B, Jinlong B or Longjing 4302B.

[0017] In the above method, the wild-type cytoplasmic male sterile line insensitive to high temperature is Zhenshan 97A, V20A, II-32A or Gang 46A.

[0018] In the above method, the corresponding maintainer line of the wild-type cytoplasmic male sterile line that is sensitive to high temperature is Zhenshan 97B, V20B, II-32B or Gang 46B.

[0019] In order to solve the above technical problems, the present invention further provides a DNA molecule, wherein the DNA molecule is a DNA fragment Rf3-nonTOS, and the nucleotide sequence of the DNA fragment Rf3-nonTOS is SEQ ID No. 2 in the sequence list.

[0020] In order to solve the above technical problems, the present invention further provides the use of the above DNA molecules or biological materials containing the DNA molecules in improving wild abortive cytoplasmic male sterile rice lines.

[0021] In the above application, the purpose of improving the wild-type cytoplasmic male sterile rice line is to make the wild-type cytoplasmic male sterile rice line with high-temperature regeneration phenotype lose its high-temperature regeneration phenotype.

[0022] In order to solve the above technical problems, the present invention also provides the use of the above DNA molecules or biological materials containing the DNA molecules in cultivating wild abortion type cytoplasmic male sterile rice lines without high temperature recovery phenotype.

[0023] In the above application, the biomaterial is any one of the following:

[0024] B1) a recombinant vector containing the DNA molecule;

[0025] B2) a recombinant microorganism containing the DNA molecule or a recombinant microorganism containing the recombinant vector described in B1);

[0026] B3) a transgenic rice cell line containing the DNA molecule, or a transgenic rice cell line containing the recombinant vector described in B1), or a transgenic rice cell line containing the recombinant microorganism described in B2);

[0027] B4) transgenic rice tissue containing the DNA molecule, or transgenic rice tissue containing the recombinant vector described in B1), or transgenic rice tissue containing the recombinant microorganism described in B2);

[0028] B5) a transgenic rice organ containing the DNA molecule, or a transgenic rice organ containing the recombinant vector described in B1), or a transgenic rice organ containing the recombinant microorganism described in B2).

[0029] In order to solve the above technical problems, the present invention further provides the use of a substance for detecting the DNA fragment Rf3-nonTOS in the genome of wild-type cytoplasmic male sterile rice in detecting whether the wild-type cytoplasmic male sterile rice has a high-temperature refertility phenotype.

[0030] In the present invention, the high temperature is ≥28°C.

[0031] The present invention provides a method for making a wild abortion type cytoplasmic male sterile rice line that is sensitive to high temperature lose its high temperature restoration phenotype, by which a DNA fragment Rf3-TOS in a wild abortion type cytoplasmic male sterile rice line with a high temperature restoration phenotype is replaced with a DNA fragment Rf3-nonTOS in a wild abortion type cytoplasmic male sterile rice line without a high temperature restoration phenotype, so that the wild abortion type cytoplasmic male sterile rice line with a high temperature restoration phenotype no longer has the high temperature restoration phenotype. The improved wild abortion type cytoplasmic male sterile rice line does not have a high temperature restoration phenotype, which lays a solid foundation for the safety of hybrid rice seed production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the breeding flow chart of Tianfeng A near-allelic line Tianfeng A-NIL.

[0033] Figure 2 This is a microscopic examination of the I2-KI staining effect of Tianfeng A and Tianfeng A-NIL pollen grains under normal temperature conditions. Figure 3 This is a microscopic examination of the I2-KI staining effect of Tianfeng A and Tianfeng A-NIL pollen grains under high temperature conditions. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1. Breeding and phenotypic identification of the wild-type cytoplasmic male sterile rice line Tianfeng A-NIL

[0037] The applicant of the present invention Figure 1The flowchart shown in the figure is used to create a wild abortive cytoplasmic male sterile rice line without a high temperature regeneration phenotype, by replacing the DNA fragment Rf3-TOS (hereinafter referred to as Rf3-TOS fragment, whose nucleotide sequence is SEQ ID No. 1 in the sequence list) in the wild abortive cytoplasmic male sterile rice line Tianfeng A (wild type, wild abortive cytoplasmic male sterile rice line sensitive to high temperature) with a wild abortive cytoplasmic male sterile rice line without a high temperature regeneration phenotype (hereinafter referred to as Rf3-nonTOS fragment, whose nucleotide sequence is SEQ ID No. 2 in the sequence list), so that the wild abortive cytoplasmic male sterile rice line Tianfeng A with a high temperature regeneration phenotype no longer has the phenotype of high temperature regeneration. The specific breeding operation steps are as follows:

[0038] 1. Use rice Zhenshan 97B (the maintainer line of rice Zhenshan 97A, i.e., the maintainer line corresponding to the wild abortion type cytoplasmic male sterile line insensitive to high temperature) as the female parent and rice Tianfeng B (the maintainer line of rice Tianfeng A, i.e., the maintainer line corresponding to the wild abortion type cytoplasmic male sterile line sensitive to high temperature) as the male parent for hybridization to obtain F1 generation seeds. Plant F1 generation seeds, use PCR amplification to detect the DNA fragments (Rf3-TOS fragments or Rf3-nonTOS fragments) carried by the F1 generation population, select F1 generation rice plants carrying the Rf3-TOS fragment, and name them Tianfeng B-Rf3-TOS-F1.

[0039] Tianfeng B-Rf3-TOS-F1 was self-pollinated to obtain F2 seeds. F2 seeds were planted, and the DNA fragments (Rf3-TOS fragments or Rf3-nonTOS fragments) carried by the F2 population were detected by PCR amplification. Combined with agronomic trait observations, 5-8 individual plants carrying Rf3-nonTOS fragments but not Rf3-TOS fragments and with plant and leaf morphology (plant height, leaf freshness, stem strength, ear color fading and fruit setting rate) similar to Tianfeng B were selected from the F2 population and named Tianfeng B-Rf3-nonTOS-F2.

[0040] Tianfeng B and Tianfeng A both contain the Rf3-TOS fragment whose nucleotide sequence is SEQ ID No. 1 in the sequence list. Zhenshan 97B and Zhenshan 97A both contain the Rf3-nonTOS fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence list.

[0041] Among them, the method of PCR amplification for detecting DNA fragments carried by rice plants is as follows: extracting DNA from individual leaves at the seedling stage, using the DNA from individual leaves as a template and Rf3-TOS-F / R as primers for PCR amplification to obtain an amplified product.

[0042] The primer sequences are as follows:

[0043] Rf3-TOS-F:5'-GCCATAAGCAACTGATACAATAG-3';

[0044] Rf3-TOS-R:5'-GGACAAACAAGCAAGAAAC-3'.

[0045] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, for a total of 35 reaction cycles; and extension at 72°C for 7 min.

[0046] The PCR amplification system was as follows: 2× Taq Plus Master Mix II 15 μl, primer Rf3-TOS-F 1 μl, primer Rf3-TOS-R 1 μl, DNA 1 μl, ddH 2 O is added to 50 μl. The DNA fragment carried by the test plant is determined according to the presence, size and sequence of the PCR product: if the PCR product of the test plant is a fragment with a nucleotide sequence of 3083-3875 of SEQ ID No.1 and a size of 793 bp, the test plant carries the Rf3-TOS fragment; if the PCR product of the test plant is not the above 793 bp fragment, the test plant carries the Rf3-nonTOS fragment but does not carry the Rf3-TOS fragment.

[0047] 2. Use Tianfeng B-Rf3-nonTOS-F2 as the female parent and Tianfeng B as the male parent for backcrossing to obtain BC1F1 generation seeds. Plant BC1F1 generation seeds, use PCR amplification (the specific method is the same as step 1) combined with agronomic trait observation to select 5-8 individual plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC1F1 generation population, and name them Rf3-TOS-BC1F1.

[0048] Rf3-TOS-BC1F1 was self-pollinated to obtain BC1F2 seeds. BC1F2 seeds were planted, and PCR amplification detection (the specific method is the same as step 1) combined with agronomic trait observation was used to select 5-8 individual plants carrying the Rf3-nonTOS fragment but not carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC1F2 population, and named them Rf3-nonTOS-BC1F2.

[0049] 3. Use Rf3-nonTOS-BC1F2 as the female parent and Tianfeng B as the male parent for backcrossing to obtain BC2F1 generation seeds. Plant the BC2F1 generation seeds, and use PCR amplification (the specific method is the same as step 1) combined with agronomic trait observation to select 5-8 individual plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC2F1 generation population, and name them Rf3-TOS-BC2F1.

[0050] Rf3TOS-BC2F1 was self-pollinated to obtain BC2F2 seeds. BC2F2 seeds were planted, and PCR amplification detection (the specific method is the same as step 1) combined with agronomic trait observation was used to select 5-8 individual plants carrying the Rf3-nonTOS fragment but not carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC2F2 population, and named them Rf3-nonTOS-BC2F2.

[0051] 4. Backcross Rf3-nonTOS-BC2F2 as the female parent and Tianfeng B as the male parent to obtain BC3F1 generation seeds. Plant BC3F1 generation seeds, use PCR amplification (specific method is the same as step 1) combined with agronomic trait observation to select 5-8 individual plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC3F1 generation population, and name them Rf3-TOS-BC3F1.

[0052] Rf3-TOS-BC3F1 was self-pollinated to obtain BC3F2 seeds. BC3F2 seeds were planted, and 5-8 individual plants carrying the Rf3-nonTOS fragment but not carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B were selected from the BC3F2 population using PCR amplification detection (the specific method is the same as step 1) combined with agronomic trait observation, and named Rf3-nonTOS-BC3F2.

[0053] 5. Backcross Rf3-nonTOS-BC3F2 as the female parent and Tianfeng B as the male parent to obtain BC4F1 generation seeds. Plant BC4F1 generation seeds, use PCR amplification (specific method is the same as step 1) combined with agronomic trait observation to select 5-8 individual plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC4F1 generation population, and name them Rf3-TOS-BC4F1.

[0054] Rf3-TOS-BC4F1 was self-pollinated to obtain BC4F2 seeds. BC4F2 seeds were planted, and PCR amplification detection (the specific method is the same as step 1) combined with agronomic trait observation was used to select 5-8 individual plants from the BC4F2 population that carried the Rf3-nonTOS fragment but did not carry the Rf3-TOS fragment and whose plant and leaf morphology was similar to that of Tianfeng B, and named them Rf3-nonTOS-BC4F2.

[0055] 6. Use Rf3-nonTOS-BC4F2 as the female parent and Tianfeng B as the male parent for backcrossing to obtain BC5F1 generation seeds. Plant BC5F1 generation seeds, use PCR amplification (the specific method is the same as step 1) combined with agronomic trait observation to select 5-8 individual plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC5F1 generation population, and name them Rf3-TOS-BC5F1.

[0056] Rf3-TOS-BC5F1 was self-pollinated to obtain BC5F2 seeds. BC5F2 seeds were planted, and PCR amplification detection (the specific method is the same as step 1) combined with agronomic trait observation was used to select 5-8 individual plants carrying the Rf3-nonTOS fragment but not carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC5F2 population, and named them Rf3-nonTOS-BC5F2.

[0057] 7. Backcross Rf3-nonTOS-BC5F2 as the female parent and Tianfeng B as the male parent to obtain BC6F1 generation seeds. Plant BC6F1 generation seeds, use PCR amplification (specific method is the same as step 1) combined with agronomic trait observation to select 5-8 individual plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC6F1 generation population, and name them Rf3-TOS-BC6F1.

[0058] Rf3-TOS-BC6F1 was self-pollinated to obtain BC6F2 seeds. BC6F2 seeds were planted, and PCR amplification detection (the specific method is the same as step 1) combined with agronomic trait observation was used to select 5-8 individual plants carrying the Rf3-nonTOS fragment but not carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng B from the BC6F2 population, and named them Tianfeng B-Rf3-nonTOS.

[0059] 8. Perform continuous self-pollination on Tianfeng B-Rf3-nonTOS. PCR amplification (the specific method is the same as step 1) combined with agronomic trait observation is used to select 5-8 individual plants that carry the Rf3-nonTOS fragment but do not carry the Rf3-TOS fragment and whose plant and leaf morphology is most similar to Tianfeng B from each self-pollination progeny for the next self-pollination. After three self-pollinations, 20 individual plants that carry the Rf3-nonTOS fragment but do not carry the Rf3-TOS fragment and whose plant and leaf morphology is similar to Tianfeng B are selected and named Tianfeng B. -改良 .

[0060] 9. Take Tianfeng A as the parent and Tianfeng B -改良 As the male parent, hybridization was performed to obtain F1 generation seeds. F1 generation seeds were planted, and 5 single plants carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng A were selected from the F1 generation population by PCR amplification (the specific method is the same as step 1) combined with agronomic trait observation, and named Tianfeng A-Rf3-TOS-F1.

[0061] 10. The obtained Tianfeng A-Rf3-TOS-F1 strain was used as the female parent and Tianfeng B -改良 As the male parent, hybridization was performed to obtain F1 generation seeds. F1 generation seeds were planted, and single plants carrying the Rf3-nonTOS fragment but not carrying the Rf3-TOS fragment and with plant and leaf morphology similar to Tianfeng A were selected from the F1 generation population by PCR amplification (the specific method is the same as step 1) combined with agronomic trait observation, and named Tianfeng A-NIL.

[0062] Example 2: Phenotypic Identification of the Wild-type Cytoplasmic Male Sterile Rice Line Tianfeng A-NIL

[0063] The wild-type Tianfeng A (control) and the Tianfeng A-NIL obtained in Example 1 (10 individual plants were randomly selected for each treatment) were subjected to normal temperature treatment (CK) and high temperature treatment using a light incubator. The normal temperature treatment conditions were: 25°C / 12h, light intensity of 30000Lx, humidity of 60%; 25°C / 12h, dark, humidity of 60%; high temperature treatment conditions were: 35°C / 12h, light intensity of 30000Lx, humidity of 60%; 28°C / 12h, dark, humidity of 60%. The mature anthers of the wild-type Tianfeng A and Tianfeng A-NIL plants were collected at the heading stage for iodine staining and microscopic examination. During iodine staining, the anthers of 3 florets of a single rice plant were placed on a glass slide, the anthers were crushed with tweezers to release the pollen grains, and 1-2 drops of iodine were added. 2-KI solution, cover with a coverslip, and observe under a microscope. All pollen grains stained blue are viable, and those stained yellow-brown are underdeveloped pollen grains (recorded as unstainable pollen grains). Three wild-type Tianfeng A (control) and Tianfeng A-NIL plants were randomly selected, and four fields of view were taken from each plant. The total number of pollen grains was greater than 500. The staining rate of pollen was calculated (the average value of the three plants was taken, and the average value was rounded to two decimal places). The staining rate was used to indicate the fertility of pollen.

[0064] The results showed that: under normal temperature conditions, there were no stainable pollen grains in the wild-type Tianfeng A and Tianfeng A-NIL plants, and the pollen iodine staining rate was 0% (see Table 1 and Figure 2 Under high temperature conditions, the wild-type Tianfeng A showed a refertility phenotype, with some dyeable pollen grains, and the pollen iodine staining rate reached 49.62%, and the anthers were fertile under microscopic examination; while the Tianfeng A-NIL treated with high temperature had no dyeable pollen grains, and the pollen iodine staining rate was 0% (see Table 1 and Figure 3 ). It can be seen that under normal temperature conditions, both the wild type Tianfeng A and Tianfeng A-NIL showed pollen abortion; under high temperature conditions, the wild type Tianfeng A showed a regeneration phenotype, while Tianfeng A-NIL did not show a regeneration phenotype, which indicates that replacing the DNA fragment Rf3-TOS in the wild abortive cytoplasmic male sterile line material with a high-temperature regeneration phenotype with the DNA fragment Rf3-nonTOS in the wild abortive cytoplasmic male sterile line material without a high-temperature regeneration phenotype can effectively overcome its high-temperature regeneration.

[0065] Table 1 Pollen grains of wild-type Tianfeng A and Tianfeng A-NIL plants 2 -KI staining results statistics

[0066]

[0067] Note: Compared with the wild type, ** represents P<0.01, which is a very significant difference.

[0068] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for causing a wild-type cytoplasmic male sterile rice line that is sensitive to high temperature to lose its high temperature restoration phenotype, comprising: Replacing the DNA fragment Rf3-TOS in the wild-type cytoplasmic male sterile rice line that is sensitive to high temperature with the DNA fragment Rf3-nonTOS, so that the high-temperature restoration phenotype of the wild-type cytoplasmic male sterile rice line that is sensitive to high temperature is lost; The nucleotide sequence of the DNA fragment Rf3-TOS is SEQ ID No. 1 in the sequence list; The nucleotide sequence of the DNA fragment Rf3-nonTOS is SEQ ID No. 2 in the sequence list; The wild-type cytoplasmic male sterile rice line that is sensitive to high temperature is a wild-type cytoplasmic male sterile rice line that has a high-temperature restoration phenotype.

2. The method according to claim 1, characterized in that: The replacement of the DNA fragment Rf3-TOS in the wild abortion type cytoplasmic male sterile rice line that is sensitive to high temperature with the DNA fragment Rf3-nonTOS is achieved by hybridizing the improved maintainer line with the wild abortion type cytoplasmic male sterile rice line that is sensitive to high temperature, and then backcrossing; The improved maintainer line is obtained by replacing the DNA fragment Rf3-TOS in the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line sensitive to high temperature with the DNA fragment Rf3-nonTOS.

3. The method according to claim 2, characterized in that: The method for replacing the DNA fragment Rf3-TOS in the maintainer rice corresponding to the wild-type cytoplasmic male sterile line sensitive to high temperature with the DNA fragment Rf3-nonTOS comprises the following steps: 1) hybridizing the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line sensitive to high temperature as the female parent and the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line insensitive to high temperature as the male parent to obtain F1 generation plants, and selfing the F1 generation plants to obtain selfing offspring; the wild abortion type cytoplasmic male sterile line insensitive to high temperature is a wild abortion type cytoplasmic male sterile line rice without high temperature re-fertilization phenotype; the wild abortion type cytoplasmic male sterile line insensitive to high temperature and the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line insensitive to high temperature both contain the DNA fragment Rf3-nonTOS; 2) Selecting a single plant carrying the DNA fragment Rf3-nonTOS but not carrying the DNA fragment Rf3-TOS from the selfed progeny obtained in step 1) as the female parent, performing backcrossing with the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line that is sensitive to high temperature as the male parent, performing continuous selfing-backcrossing, and then continuous selfing, selecting a single plant carrying the DNA fragment Rf3-nonTOS but not carrying the DNA fragment Rf3-TOS from the selfed progeny to obtain an improved maintainer line, thereby completing the replacement of the DNA fragment Rf3-TOS in the maintainer rice corresponding to the wild abortion type cytoplasmic male sterile line that is sensitive to high temperature with the DNA fragment Rf3-nonTOS.

4. The method according to claim 3, characterized in that: In the above 2), the number of consecutive self-crossing and backcrossing is at least 5 times, and the backcrossing mother in the self-crossing and backcrossing is a parent that carries the DNA fragment Rf3-nonTOS but does not carry the DNA fragment Rf3- Individual strains of TOS.

5. The method according to claim 3 or 4, characterized in that: In the above 2), the number of consecutive self-pollinations is at least 3 times, and the selfing parent is a single plant that carries the DNA fragment Rf3-nonTOS but does not carry the DNA fragment Rf3-TOS.

6. The method according to any one of claims 1 to 5, characterized in that: The wild-type cytoplasmic male sterile line that is sensitive to high temperature is Tianfeng A, Wufeng A, Jinlong A or Longjing 4302A; And / or, the corresponding maintainer line of the wild-type cytoplasmic male sterile line that is sensitive to high temperature is Tianfeng B, Wufeng B, Jinlong B or Longjing 4302B; And / or, the maintainer line corresponding to the wild-type cytoplasmic male sterile line insensitive to high temperature is Zhenshan 97B, V20B, II-32B or Gang 46B.

7. A DNA molecule, characterized in that: The DNA molecule is a DNA fragment Rf3-nonTOS, and the nucleotide sequence of the DNA fragment Rf3-nonTOS is SEQ ID No. 2 in the sequence list.

8. Use of the DNA molecule or biological material containing the DNA molecule as claimed in claim 7 in improving wild-type cytoplasmic male sterile rice.

9. Use of the DNA molecule or biological material containing the DNA molecule as claimed in claim 7 in cultivating wild-type cytoplasmic male sterile rice lines without high-temperature regeneration phenotype.

10. Application of the substance of detecting the DNA fragment Rf3-nonTOS in the genome of wild-type cytoplasmic male sterile rice to detect whether the wild-type cytoplasmic male sterile rice has a high-temperature regeneration phenotype; The nucleotide sequence of the DNA fragment Rf3-nonTOS is SEQ ID No. 2 in the sequence listing.

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