CN112586341B - Preparation method of recovery line japonica rice in late flowering - Google Patents
Preparation method of recovery line japonica rice in late flowering Download PDFInfo
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Abstract
The invention provides a preparation method of restoring japonica rice in late flowering, which comprises the following steps: planting the backcross first-generation seeds to obtain second-generation plants; in the second-generation plants, screening out plants which have the flowering time 11:00 in the morning and have the restorer gene Rf1a, and carrying out backcross on the plants and the japonica rice to obtain backcross second-generation seeds; planting the second generation seeds of the backcross seeds to obtain second generation plants of the backcross seeds; screening out plants which have the restoring gene Rf1a and have the flowering time of 11:00 in the morning from the second generation plants of the backcross son for cultivation to obtain first generation seeds of the backcross son; and (4) adding generations of the first generation of self-crossed seeds until the characters are stable, and obtaining the restoring line japonica rice when the rice is late-flowering. The restoring line japonica rice obtained by the preparation method has late flowering time and can improve pollination and maturing rate of hybrid japonica rice.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a preparation method of japonica rice of a late flowering restoring line.
Background
Rice is one of the most important food crops in the world, and more than half of the population in the world uses rice as staple food, so that the yield of rice is related to the food safety in the world. The utilization of heterosis is an effective way to improve the yield of rice, and the development of Chinese hybrid indica rice has achieved great success, but the development of hybrid japonica rice is slow.
The low seed yield is an important bottleneck for restricting the development of the hybrid japonica rice. At present, a japonica rice sterile line and an indica-japonica hybrid restoring line are respectively used as parents to prepare hybrid japonica rice, but the dissimilarity of the florescence of the parents is a key factor causing the low yield of the hybrid japonica rice seeds. Specifically, indica rice blooms early, japonica rice blooms late, and japonica rice sterile line is directly transferred from japonica rice, and sterile cytoplasm effect is added, so that the japonica rice blooms late, usually after 11: 00; the flowering time of the indica-japonica hybrid restorer is usually 10: 00-11: and when the hybrid japonica rice is bloomed between 30 days, namely after the indica japonica hybrid restorer bloomed to loose powder, the japonica rice sterile line bloomes in the coming late, so that the flowering time of the indica japonica hybrid restorer and the japonica rice sterile line is only overlapped for half an hour, and the indica japonica hybrid restorer and the japonica rice sterile line are used as parents for hybridization, so that the pollination and seed setting rate of the japonica rice sterile line on the day of flowering is low, the general pollination and seed setting rate is about 20%, the seed yield is low, and the development of hybrid japonica rice is restricted.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of the restoring line japonica rice in late flowering, and the restoring line japonica rice in late flowering obtained by the preparation method has the advantages of late flowering time and capability of improving pollination and seed setting rate of hybrid japonica rice.
The embodiment of the invention provides a preparation method of restoring japonica rice in late flowering, which comprises the following steps:
hybridizing indica rice and japonica rice which have the restoring gene Rf1a and have the flowering time of 11:00 in the morning to obtain a first filial generation seed;
planting the first filial generation seeds to obtain first filial generation plants;
backcrossing the first filial generation plant with the japonica rice to obtain a first backcross seed with the restoring gene Rf1 a;
planting the backcross first-generation seeds to obtain second-generation plants;
in the second-generation plants, screening out plants which have the flowering time 11:00 in the morning and have the restorer gene Rf1a, and carrying out backcross on the plants and the japonica rice to obtain backcross second-generation seeds;
planting the second generation seeds of the backcross seeds to obtain second generation plants of the backcross seeds;
screening out plants which have the restoring gene Rf1a and have the flowering time of 11:00 in the morning from the second generation plants of the backcross son for cultivation to obtain first generation seeds of the backcross son;
and (4) adding generations of the first generation of self-crossed seeds until the characters are stable, and obtaining the restoring line japonica rice when the rice is late-flowering.
Specifically, the japonica rice is japonica rice R19.
Specifically, among the third generation plants with the fruit set rate of more than 90%, plants having the restorer gene Rf1a and flowering time 11:00 in the morning are selected for cultivation.
Specifically, a plant having a restorer gene Rf1a at 11:00 am after flowering is selected, and offspring having stable traits are obtained by line breeding.
Specifically, the generation of the first generation of self-cross seeds is added to the fourth generation of self-cross seeds, the fourth generation of self-cross seeds is matched with sterile japonica rice, the fourth generation of self-cross seeds with stable characters is screened, namely the late-flowering restoring line japonica rice, and the conditions for screening the fourth generation of self-cross seeds with stable characters comprise: a. in the combination, the maturing rate of the four generations of plants of the self-cross seeds, which accounts for more than 80% of the total number, reaches more than 90%; b. in the combination, the yield of the four generations of plants of the auto-crossed seeds, which accounts for more than 60% of the total number, is more than 5% higher than that of the indica rice or the japonica rice; c. in the combination, the rice quality of the four generations of plants of the self-cross seeds, which accounts for more than 50 percent of the total number, reaches the national standard grade 2.
Further, sterile line japonica rice transferred from main cultivars in japonica rice regions of northeast early japonica middle maturity type region, northern early japonica late maturity type region, northern China middle japonica middle maturity type region, Yangtze river middle and downstream single season late japonica type region and southwest plateau wet single season rice cropping region is selected for testing and matching.
Further, the restorer gene Rf1a in the plant with stable character is retested, and the plant with stable character containing the restorer gene Rf1a is used as the restoring line japonica rice at the time of late flowering.
Specifically, in the plants with stable characters, progeny with the late-flowering character is screened out by using a molecular marker, and is backcrossed with the japonica rice, and the method specifically comprises the following steps: the plant with stable characters to be detected is amplified by adopting a forward primer shown as SEQ ID NO. 1 in the sequence table and a reverse primer shown as SEQ ID NO. 2 in the sequence table.
Specifically, the trait is stable to a consistent agronomic trait of two years.
Specifically, the second generation of seeds is obtained by backcrossing japonica rice with the screened plants which have the agronomic characters similar to japonica rice and the flowering time 11:00 in the morning and have the restoring gene Rf1 a.
Specifically, the self-mating first-generation seeds are subjected to additional generation until the properties are stable, and the heading period temperature and the sunlight condition are simulated to obtain the restoring line japonica rice at the late flowering time, wherein the heading period temperature and sunlight condition simulation method comprises the following steps: adopting an artificial climate box, and respectively setting 5 groups of test parameters, wherein the first group of test parameters is as follows: turning on the light when 15 days, turning off the light when 9 days are next, and keeping the temperature at 27 ℃; the second set of test parameters is: turning on illumination at 14 ℃ and turning off illumination at 10 ℃ on the second day, and keeping the temperature at 30 ℃; the third set of test parameters is: turning on illumination when 13.5 days, turning off illumination when 10.5 days are next days, and keeping the temperature at 32 ℃; a fourth set of the test parameters is: turning on illumination when 13 days, turning off illumination when 11 days next, and keeping the temperature at 34 ℃; the fifth set of test parameters was: and starting illumination at 13 days, closing illumination at 11 days, keeping the temperature at 27 ℃, and selecting plants with flowering time concentrated between 11:00 and 13:30 under the five groups of test parameters as the late-flowering restoring line japonica rice.
The embodiment of the invention provides a preparation method of japonica rice of a late flowering time restorer line, the flowering time of the japonica rice of the late flowering time restorer line obtained through the preparation method is 10: 30-12: 00, and compared with the existing japonica rice restorer line, the flowering time is obviously delayed, so that the flowering time of the japonica rice of the late flowering time restorer line provided by the embodiment of the invention can be basically synchronous with the flowering time of the japonica rice sterile line hybrid, the japonica rice of the late flowering time restorer line can be fully pollinated on the same flowering day, in addition, the pollen quantity of the japonica rice of the late flowering time restorer line provided by the embodiment of the invention can be increased, the seed production and seed setting rate can reach 60%, and the seed yield is greatly increased.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.
FIG. 1 shows the amplification results of an example of the present invention;
fig. 2 is a comparison graph of progeny obtained by hybridizing the late-flowering restorer japonica rice (R19) as a male parent with the japonica rice sterile line (L6A) and progeny obtained by hybridizing the japonica rice restorer line (C418) as a male parent with the japonica rice sterile line (L6A) in the embodiment of the present invention, in which the male parent of the left rice is japonica rice restorer line (C418) and the male parent of the right rice is late-flowering restorer japonica rice (R19).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Examples
The embodiment of the invention provides a preparation method of restoring japonica rice in late flowering, which comprises the following steps:
hybridizing indica rice and japonica rice which have the restoring gene Rf1a and have the flowering time of 11:00 in the morning to obtain first filial generation seeds; in this embodiment, the indica rice is also required to have the characteristic of good appearance and quality; the selected japonica rice needs to have the characteristics of rice quality over grade 2, sufficient pollen quantity, good yield, strong rice blast resistance and strong general combining ability, and when the japonica rice is realized, IR8 is selected as indica rice used in the embodiment of the invention, and R313 is selected as japonica rice used in the embodiment of the invention.
Planting first filial generation (F)1) Seeds to obtain a first filial generation plant; in this example, the number of first filial generation seeds obtained was 500 or more, and 50 of the first filial generation seeds were planted.
Backcrossing the first filial generation plant with japonica rice R313 to obtain the first Backcross (BC) generation with the restoring gene Rf1a1F1) Seeds; in this example, the backcross first generation seed with 1145bp characteristic band is regarded as having the restorer gene Rf1a, and when it is implemented, it can be detected by the existing molecular marker method, specifically: the forward primer shown as SEQ ID NO. 1 in the sequence table and the reverse primer shown as SEQ ID NO. 2 in the sequence table are adopted to amplify the backcross first generation (BC) to be detected1F1) Seeds, the amplification steps are as follows: the annealing temperature is 54 ℃, the target band length is 1145bp, if the backcross first generation (BC) to be detected1F1) The seeds containing the desired band are identified as having the restorer gene Rf1a, and the first backcross generation (BC) to be tested1F1) When the amplified band of the seed is 571bp, it is determined that the restorer gene Rf1a is not contained.
An amplification system: 5 microliter of PCR premix, 3.6 microliter of dd water, 0.2 microliter of each of the upstream primer and the downstream primer, 1 microliter of DNA template, and 10 microliter of amplification system in total.
Planting backcross first generation (BC)1F1) Seeds to obtain the second generation plants; selecting japonica rice with agronomic character in the second generation plant, wherein the flowering time is 11:00 in the morning and the recovery is realizedThe gene Rf1a and plants with excellent comprehensive properties are backcrossed with japonica rice R313 to obtain the second-generation seeds with the restorer gene Rf1a, and in the embodiment, the obtained second-generation seeds are more than 500 seeds, and 50 backcrossed second-generation seeds are planted. The plants of the japonica rice with the agronomic characters are rich in genetic components of the japonica rice, the screened plants of the japonica rice with the agronomic characters can ensure that the seed setting rate of the second generation seeds of the recurrent rice is higher and the recovery spectrum is wider, and the agronomic characters comprise: dense glumes, green leaves, dense color, smooth leaves, short and straight leaves, compact plant type, difficult falling, and the like. In this example, the comprehensive character means that the rice quality is excellent, the pollen quantity is large, the yield is good, the rice blast resistance is strong, and the combining ability is strong.
In this example, if the setting percentage is less than 90%, the second generation of plants can be selected from good single plants with rice quality above international grade 2, large pollen quantity, good yield, strong rice blast resistance and strong combining ability, and the good single plants are backcrossed with japonica rice R313 once again until the setting percentage reaches more than 90%. Wherein, high yielding nature includes well: big ear (180 grains/ear or more) and middle tiller (15 grains/acupoint).
Planting the second generation seeds of the backcross seeds to obtain second generation plants of the backcross seeds;
among the second generation plants of the backcross, plants having a flowering time 11:00 am and a restorer gene Rf1a were selected and added to the fourth generation (BC) of the inbred by the line method2F4) Obtaining progeny seeds with stable characters;
planting of the fourth generation of the self-mating seed (BC)2F4) The seeds are planted by adopting single seedlings for more than 300 plants, the agronomic characters required by flowering time and DUS national standard are identified, the performances are consistent, and the progeny has no separation phenomenon. More than 50 strains were randomly selected and retested with Rf1 a. The agronomic character expression and the recovery gene detection in the field are consistent in two years, namely the agronomic character is stable.
Meanwhile, the combining ability of the plants with stable characters is measured, specifically, the combining ability of the plants with stable characters and more than 20 japonica rice sterile lines in the northeast rice region or the northeast rice region is measured, the general combining ability (gca) of the plants with stable characters is measured, and the general combining ability is measured to be 9.58. Specifically, the conditions for screening plants with stable characters comprise: a. more than 80% of plants with stable characters have the setting percentage of more than 90%; b. the yield of more than 60% of plants with stable characters is more than 5% higher than that of parents; c. more than 50% of plants with stable characters have rice quality reaching the national standard grade 2.
Specifically, among the progeny with stable characters, the progeny with the late-flowering character is screened out by using a molecular marker to be backcrossed with japonica rice R313, and the detection can be carried out by using the existing molecular marker method, specifically: adopting a forward primer shown as SEQ ID NO. 1 in the sequence table and a reverse primer shown as SEQ ID NO. 2 in the sequence table to amplify the offspring with stable characters to be detected, wherein the amplification steps are as follows: the annealing temperature was 54 ℃ and the target band length was 1145bp, and the late flowering trait was identified as having the stable trait to be tested if the target band was contained in the offspring, and the late flowering trait was not identified if the amplified band of the stable trait to be tested was 571 bp. The amplification results of this example are shown in FIG. 1.
The amplification system comprises: 5 microliter of PCR premix, 3.6 microliter of dd water, 0.2 microliter of each of the upstream primer and the downstream primer, 1 microliter of DNA template, and 10 microliter of amplification system in total.
Specifically, a plant having a restorer gene Rf1a at 11:00 am after flowering is selected, and offspring having stable traits are obtained by line breeding. In this example, the strain method comprises the following specific steps: planting BC2F1Seeds, more than 500 colonies are constructed, plants which bloom before 11 am or have the height obviously inconsistent with the shape of plant leaves and the like before are eliminated through field investigation, and BC is harvested after maturation2F2And (4) seeds. Hybrid BC2F2Seeds, constructing more than 500 colonies, repeating the steps, eliminating the plants which blossom or have the height which is obviously inconsistent with the shape of the plant leaves and the like before 11 am by field observation, and harvesting the BC after maturation2F3And (4) seeds. Until adding to BC2F4。
Specifically, after six generations of plants are bred until the characters are stable, the restorer gene Rf1a is retested, and the plant with the restorer gene Rf1a is used as the late-flowering restoring japonica rice.
Specifically, the trait is stable to be consistent with the agronomic trait for two years. In this embodiment, the agronomic property includes flowering time.
Specifically, the method for simulating the heading stage temperature and the sunshine condition in the main production area of japonica rice in China comprises the following steps: adopt the artificial climate case, set up 5 groups of test parameters respectively, first group's test parameter is: turning on the light when 15 days, turning off the light when 9 days are next, and keeping the temperature at 27 ℃; the second set of test parameters was: turning on illumination at 14 ℃ and turning off illumination at 10 ℃ on the second day, and keeping the temperature at 30 ℃; the third set of test parameters was: turning on illumination when 13.5 days, turning off illumination when 10.5 days are next days, and keeping the temperature at 32 ℃; the fourth set of test parameters was: turning on illumination when 13 days, turning off illumination when 11 days next, and keeping the temperature at 34 ℃; the fifth set of test parameters was: and starting illumination at 13 days, closing illumination at 11 days, keeping the temperature at 27 ℃, and selecting plants with flowering time concentrated between 11:00 and 13:30 under the five groups of test parameters as the recovery line japonica rice in late flowering. The specific setup is shown in table 1.
Table 1 shows 5 sets of test parameters
Note: the temperatures in table 1 refer to the temperatures at light and dark.
The comparison results of the late-flowering restorer line japonica rice (R19) obtained in the example of the present invention as a male parent hybridized with the japonica rice sterile line (L6A) and the japonica rice restorer line (C418) as a male parent hybridized with the japonica rice sterile line (L6A) as a control are shown in table 2.
Table 2 shows two control groups
As can be seen from table 2, the flowering time of the japonica rice of the late-flowering restorer line provided in the embodiment of the present invention is 10:30 to 12:00, and the flowering time is significantly delayed compared to the japonica rice of the restorer line of the control (C418), so that the flowering time of the japonica rice of the late-flowering restorer line provided in the embodiment of the present invention can be substantially synchronized with the flowering time of the japonica rice hybrid of the sterile line, and the flowering time of the sterile line (L6A) is 10:30 to 12:00, so that the half-hour flowering time overlap can be achieved on the day of flowering of the japonica rice of the late-flowering restorer line, which enables the japonica rice of the sterile line and the japonica rice of the restorer line to be fully pollinated, and the amount of pollen of the japonica rice of the restorer line provided in the embodiment of the present invention can be increased, which enables the maturing rate to be up to 60%, as can also be seen from fig. 2, the maturing rate of the japonica rice of the late-flowering restorer line provided in the embodiment of the present invention can be significantly increased over the control, meanwhile, the obtained progeny rice quality can reach the national standard grade 1, and the rice blast resistance is good.
Although the embodiments of the present invention have been described above, the above description is only for the convenience of understanding the present invention, and is not intended to limit the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (8)
1. A preparation method of japonica rice of a recovery line when late flowering is characterized by comprising the following steps:
hybridizing indica rice which has the flowering time of 11:00 in the morning and has the restorer gene Rf1a with japonica rice to obtain first filial generation seeds, selecting IR8 as the indica rice and R313 as the japonica rice;
planting the first filial generation seeds to obtain first filial generation plants;
backcrossing the first filial generation plant with the japonica rice to obtain a first backcross seed with the restoring gene Rf1 a;
planting the backcross first-generation seeds to obtain second-generation plants;
screening plants which have agronomic characters similar to japonica rice and flowering time 11:00 in the morning and have the restoring gene Rf1a from the second filial generation plants to carry out backcross with the japonica rice to obtain backcross second filial generation seeds;
planting the second generation seeds of the backcross seeds to obtain second generation plants of the backcross seeds;
screening out plants which have the restoring gene Rf1a and have the flowering time of 11:00 in the morning from the second generation plants of the backcross son for cultivation to obtain first generation seeds of the backcross son;
and (4) adding generations of the first generation of self-crossed seeds until the characters are stable, and obtaining the restoring line japonica rice when the rice is late-flowering.
2. The method according to claim 1, wherein a plant having a restorer gene Rf1a is selected from the second generation plants of the backcross which have a fruiting rate of 90% or more and flowering time 11:00 am.
3. The method according to claim 1, wherein a plant having a restorer gene Rf1a is selected from the plants of the second generation of the backcross progeny, and the plants having a flowering time 11:00 am are bred by a line breeding method to obtain progeny having stable traits.
4. The method according to claim 1, wherein the generation of the first generation of self-crossed seeds is added to the fourth generation of self-crossed plants, the fourth generation of self-crossed plants is matched with sterile japonica rice, and the fourth generation of self-crossed plants with stable properties is selected as the late-flowering restoring line japonica rice, and the conditions for selecting the fourth generation of self-crossed plants with stable properties include: a. in the combination, the maturing rate of the four generations of plants of the self-cross seeds, which accounts for more than 80% of the total number, reaches more than 90%; b. in the combination, the yield of the four generations of plants of the auto-crossed seeds, which accounts for more than 60% of the total number, is more than 5% higher than that of the indica rice or the japonica rice; c. in the combination, the rice quality of the four generations of plants of the self-cross seeds, which accounts for more than 50 percent of the total number, reaches the national standard grade 2.
5. The method according to claim 4, wherein sterile japonica rice of the main cultivar-transferred sterile japonica rice in japonica rice regions of the northeast early japonica middle-maturing type region, the northern early japonica late-maturing type region, the northern mid-japonica middle-maturing type region, the southern Yangtze single-season late-maturing type region, and the southern west high wet single-season rice growing region is selected for testing and matching.
6. The method according to claim 4, wherein the restorer gene Rf1a in the plant having stable trait is retested, and the plant having stable trait and containing the restorer gene Rf1a is used as the late-flowering restorer japonica rice.
7. The method according to claim 1, wherein progeny having a late-flowering trait are selected from plants having stable traits by using a molecular marker, and backcrossed with japonica rice, the method comprising: the plant with stable characters to be detected is amplified by adopting a forward primer shown as SEQ ID NO. 1 in the sequence table and a reverse primer shown as SEQ ID NO. 2 in the sequence table.
8. The method according to claim 1, wherein the generation of the self-mating first-generation seeds is added to stable characters, and heading-stage temperature and sunlight conditions are simulated to obtain the restoring-line japonica rice at late flowering, and the heading-stage temperature and sunlight conditions simulation method comprises the following steps: adopting an artificial climate box, and respectively setting 5 groups of test parameters, wherein the first group of test parameters is as follows: turning on the light when 15 days, turning off the light when 9 days are next, and keeping the temperature at 27 ℃; the second set of test parameters is: turning on illumination at 14 ℃ and turning off illumination at 10 ℃ on the second day, and keeping the temperature at 30 ℃; the third set of test parameters is: turning on illumination when 13.5 days, turning off illumination when 10.5 days are next days, and keeping the temperature at 32 ℃; a fourth set of the test parameters is: turning on illumination when 13 days, turning off illumination when 11 days next, and keeping the temperature at 34 ℃; the fifth set of test parameters was: and starting illumination at 13 days, closing illumination at 11 days, keeping the temperature at 27 ℃, and selecting plants with flowering time concentrated between 11:00 and 13:30 under the five groups of test parameters as the late-flowering restoring line japonica rice.
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