CN112314429B - Breeding method of rice nuclear male sterility maintainer line - Google Patents
Breeding method of rice nuclear male sterility maintainer line Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a breeding method of a rice nuclear male sterile maintainer line, in particular to a rice nuclear male sterile maintainer line created by combining hybridization, molecular markers and phenotypic selection (plant leaf morphology, outcrossing fructification, bentazon sensitivity, bispyribac-sodium resistance, pollen fertility, fluorescence and the like). The breeding method provided by the invention can be used for breeding the rice nuclear male sterile maintainer line in batch and on a large scale, and further breeding the rice nuclear male sterile line, so that the individual requirements of rice production in different regions are met, and the germplasm resource utilization rate and the breeding efficiency are greatly improved.
Description
Technical Field
The invention relates to the technical field of creation of a nuclear male sterile maintainer line of a crop, in particular to creation of a nuclear male sterile maintainer line of rice by combining hybridization, molecular marking and phenotype selection of rice.
Background
Rice is one of the most important food crops in China, and more than half of the population in China takes rice as staple food. The three-line sterile line of rice has good sterility and fertility stability, is applied to hybrid rice breeding by breeders firstly, and successfully realizes the purpose of leaping again after the rice single yield of China is followed by the dwarf breeding. However, the utilization of the three lines has the restriction of recovering and preserving relationship, and the matching is not free, so that the wide utilization of germplasm resources is difficult to realize, and the strong dominant hybrid combination is difficult to screen and obtain (Yuan Longping. The third generation hybrid rice preliminary research success. Scientific report 2016, 61 (31): 3404).
Two lines based on photo-thermo genic male sterility also have some inherent problems, and the propagation of two lines of seeds and the production of hybrid seeds both require strict environmental conditions and are susceptible to unpredictable environmental changes. In addition, the critical temperature for fertility transformation of two lines will rise from generation to generation after several generations of propagation. The critical temperature trait of fertility transformation is influenced by genetic background, significantly increasing the difficulty and uncertainty of breeding new, practical, two lines (Chen L, lei D (2011) thoroughness and practice on body proposals beyond research and application of two-line hybrid. Chin J Rice Sci 18 (2): 79-85).
Nuclear male sterility is a ubiquitous phenomenon that has been found in a wide variety of plants. The first report of the phenomenon of nuclear male sterility of rice in China was the "male sterility of rice" published in 1966 by Mr. Yuan Longping. The nuclear male sterile line has many advantages, such as stable sterility and free matching, and overcomes the defects of the three-line sterile line restricted by restoration and preservation relationship, the non-free matching and the two-line sterile line affected by light and temperature conditions, such as 'beat' (Yuan Longping, the success of the preliminary research of the third generation hybrid rice, scientific report 2016, 61 (31): 3404), and is an ideal basic material for the heterosis utilization of rice, but the mass propagation problem of the nuclear male sterile line limits the production and application thereof.
In 1993, belgium PLANT GENETIC SYSTEM provided that a maintainer line of a male sterile plant can be obtained by introducing three sets of elements, namely a fertility restorer gene, a pollen lethal gene and a reporter gene (such as a fluorescent protein gene) which are expressed in a close linkage manner into a homozygous recessive nuclear male sterile plant, so as to realize the propagation of the sterile line and the maintainer line. Subsequently, perez-Prat et al (2002) further suggested that maintainer lines of male-sterile plants can be obtained by introducing two sets of elements for linkage expression, namely a fertility restorer gene and a reporter gene for selection, into homozygous male-sterile plants. In 2006, du pont pioneer in the united states takes the initiative of the above three-element technology concept to implement a Seed production technology based on nuclear male sterile mutant material in corn, and formally named the technology as SPT (Seed production technology) technology. In hybrid rice breeding, mr. Yuan Longping is referred to as "3 rd generation hybrid rice breeding technique".
At present, two research groups in China have respectively obtained a nuclear male sterility maintainer line with Huanghua occupation and 9311 as genetic backgrounds by using genetic engineering techniques (Chang, et al (2016) Construction of a large sterility system for hybrid and a seed production using a nuclear male sterility gene. PNAS,113 (49) 14145-14150 Song, et al (2020) A novel Construction for a cloning a new system of a nuclear sterility gene and a genetic male sterility gene. Plant J., https:// doi. Organ/8978 z 8978/pbi.13457).
Disclosure of Invention
In the prior art, a transformation receptor is used to obtain a rice nuclear male sterile maintainer line by a genetic engineering means, and the nuclear male sterile line and the maintainer line are bred by self-crossing of the maintainer line, wherein the transformation receptor material such as Zhonghua 11 and the like generally has better genetic transformation efficiency, but the transformation receptor does not have the characteristics required by a sterile line/a maintainer line, such as good flowering habit, high outcrossing maturing and the like.
In order to overcome the technical defects in the prior art, the first object of the invention is to provide a breeding method of a rice nuclear male sterile maintainer line with good and stable phenotype. The technical route of the breeding method is shown in figure 1, and the breeding method comprises the following steps:
(1) Selecting rice nuclear male sterility intermediate maintainer with genotype aTt and carrier element as female parent, selecting rice material with excellent agronomic character as male parent, and hybridizing to obtain F 1 Seed generation;
(2) To F 1 Selecting generation plants, keeping fertile plants with the genotype of AaTt, selecting single plants with excellent agronomic characters in the heading stage, performing fluorescence color selection on mature seeds, and keeping fluorescent seeds;
(3) To F 2 Selecting generation plants, reserving fertile plants with the genotype of aaTt, selecting single plants with excellent agronomic characters in the heading stage, performing fluorescence color selection on mature seeds, and reserving fluorescent seeds;
(4) Selection of F 3 To F n(n≥5) In the generation plants, the fluorescent seeds generated by the single plants with excellent agronomic characters in the heading stage are nuclear male sterileBreeding a maintainer line, and selecting non-fluorescent seeds generated by a single plant with excellent agronomic characters in a heading period as a nuclear male sterile line;
wherein A represents a nuclear male sterility locus wild-type allele, and a represents a nuclear male sterility allele; t represents the presence of a carrier element and T represents the absence of a carrier element.
The "rice nuclear male sterility intermediate maintainer line" and "nuclear male sterility intermediate maintainer line" in the present invention refer to a rice nuclear male sterility maintainer line to be improved.
The screening method of the female parent in the step (1) comprises the following steps:
(1) a seed stage: selecting about 100 dry seeds from each single plant of the mature nuclear male sterile intermediate maintainer line by using a fluorescence microscope or a color sorter, and carrying out fluorescence color sorting on the remaining seeds for later use; eliminating single seeds with unobvious luminescence of the fluorescent seeds, and sowing the single fluorescent seeds and the standby seeds with obvious luminescence of the fluorescent seeds and 1:1 ratio of the fluorescent seeds to the non-fluorescent seeds;
(2) seedling stage: three sets of screening elements, namely a fluorescent screening element, a bentazon sensitive element and a bispyribac-sodium resistant element, are closely linked to the carrier element T. Therefore, plants carrying the transgenic elements will exhibit sensitivity to bentazon and high resistance to herbicides such as bispyribac-sodium.
In the 3-4 leaf stage of the fluorescent seed seedlings, a commercial herbicide containing bentazon or similar chemical action components is used as a screening agent for spraying, plants carrying transgenic elements show herbicide sensitivity to cause death, and the fact that the bentazon sensitive elements work normally in the transgenic plants is verified. The method comprises the steps of spraying herbicide such as bispyribac-sodium as a screening agent in the 2-3 leaf stage of the seedling of the standby seed, killing non-transgenic plants, and showing high resistance of the herbicide such as bispyribac-sodium due to the fact that the transgenic plants carry the bispyribac-sodium resistance gene. Then transplanting the surviving spare seed seedlings.
(3) And (3) booting: selecting excellent single plants with short plant height-medium, compact plant type, erect sword leaves, medium tillering-multiple stems and strong and/or good toughness, taking leaves, extracting DNA, carrying out vector element identification by utilizing PCR, and keeping the single plants with each element existing to continue the next test.
(4) And (3) heading stage: and (3) determining that each element has a single plant through PCR detection at the booting stage, continuously performing phenotype selection, selecting the single plant with high stigma exposure and lighter neck wrap, taking mature glumous flowers, performing pollen iodine staining, and reserving the single plant with the ratio of sterile pollen to fertile pollen of 1:1 for later use.
By adopting the female parent screening method, the screened female parent has the advantages of compact plant type, erect flag leaves, strong stems, good toughness and the like; and the genotype of the selected female parent single plant can be visually judged whether to contain the complete carrier element T and whether the functional expression of the element T is complete or not from the phenotype by the modes of fluorescent color selection, bispyribac-sodium spraying, bentazone spraying, pollen iodine staining and the like, so that the breeding speed is favorably improved.
The female parent in the step (1) can be a nuclear male sterile intermediate maintainer line with sterile and fertile pollen proportion of 1:1, bentazon sensitivity, bispyribac-sodium resistance, comprehensive excellent agronomic characters and genotype of aaTt.
The male parent in the step (1) can be a nuclear male sterile hybrid line (with the genotype of Aatt), a nuclear male sterile intermediate maintainer line (with the genotype of Aatt), a three-line maintainer line (with the genotype of AAtt), a two-line sterile line (with the genotype of AAtt) and conventional rice (with the genotype of AAtt) with the characteristic characteristics of a sterile line.
The functional elements in the step (1) comprise a fertility restorer element, a pollen lethal element and/or a screening marker element which are closely linked; preferably, the screening marker element is a fluorescent protein element, a bentazon sensitive element, a bispyribac-sodium resistant element and the like.
The screening of step (2) comprises, at F 1 And (3) spraying bispyribac-sodium to kill non-transgenic plants in the seedling stage of 2-3 leaves.
The excellent agronomic traits in steps (2) - (3) include: high stigma exposure, light wrapped neck, compact plant type, erect sword leaf, more tillers, strong and/or good toughness of stem, high seed setting rate, good lodging resistance, excellent rice quality, and one or more characters of 1:1 ratio of sterile and fertile pollen.
In the step (4), the outcrossing fructification of the nuclear male sterile line with basically stable phenotype at high temperature needs to be examined, and the method comprises the following steps:
a) According to the growth period condition, F obtained by screening n-2(n≥5) To F n(n≥5) The nuclear male sterile maintainer line and the sterile line are sown in batches, so that the heading period of all planting materials is concentrated in the months with higher temperature in Hainan, such as 5-7 months, and the daily maximum temperature is 34-38 ℃; wherein F is derived from the same individual n-2(n≥5) To F n(n≥5) The nuclear male sterile line is properly sown for several days earlier than the corresponding nuclear male sterile maintainer line, and the two lines are guaranteed to shoot at the same time or the nuclear male sterile line shoots for about 1 day earlier;
b) F from the same individual plant at the time of transplantation n-2(n≥5) To F n(n≥5) The nuclear male sterile line and the maintainer line are planted according to 49 plants in 7 rows by 7 columns, the nuclear male sterile line is respectively planted at the positions of 3 rows and 4 columns, 4 rows and 3 columns, 4 rows and 4 columns, 4 rows and 5 columns and 5 rows and 4 columns, the other positions are all planted with the nuclear male sterile maintainer line from the same single plant, and the powder is removed every day when the ear is pulled and is trimmed;
c) After the seeds are yellow and ripe, the fructification condition is observed by naked eyes, the seed fructification of the nuclear male sterile line is kept as a middle and upper population, and the population with low fructification rate is eliminated.
Through the screening of the steps, the nuclear male sterile maintainer line which can still keep better outcrossing fruiting at high temperature can be obtained, and a solid foundation is laid for finally obtaining the nuclear male sterile line which is high temperature resistant and high in outcrossing fruiting.
Another object of the present invention is to provide a method for mass propagation of nuclear male sterile lines. The nuclear male sterile maintainer line with excellent characters and stable phenotype obtained by the breeding method is inbred, the inbred seeds are subjected to fluorescence color selection, and the non-fluorescence seeds are the nuclear male sterile line.
Based on the understanding of the skilled person in the art, the method for breeding the stable rice nuclear male sterile maintainer line or the method for propagating the nuclear male sterile line in batches of the invention to screen the dominant rice hybrid combination also belongs to the protection scope of the invention.
Compared with the existing method for creating the nuclear male sterile maintainer line, the method has the following advantages and effects:
1. breaks through the situation that only the nuclear male sterility maintainer line with single genetic background can be created by the transgenic technology at present.
2. More screening marker elements (such as bentazon sensitive elements and bispyribac-sodium resistant elements) are added, so that a breeder can screen and obtain required plants only through simple screening, such as herbicide spraying, in the process of creating the nuclear male sterility maintainer line, and the breeding selection efficiency is greatly improved.
3. At F 1 In the selection of the male parent and the female parent of the cross, germplasm resources with excellent genetic background and relatively excellent comprehensive characters can be selected as parent and female parent materials, and through strong combination, more excellent characters can be more easily polymerized by offspring, and the probability of obtaining a strong dominant nuclear male sterility maintainer line by screening is greatly improved.
4. Compared with the prior art, the whole technical route relates to the carrier element molecular marker verification, the carrier element phenotype screening, the multi-generation plant height, the lodging resistance, the plant leaf morphology, the outcrossing habit and other phenotype character tracing and investigation, and the nuclear male sterile maintainer line which meets the rice production requirement can be finally obtained under the organic combination of the modern molecular marker technology and the conventional breeding technology.
Drawings
FIG. 1 is the technological line of breeding male sterile maintainer line.
FIG. 2 is a comparison of the seeds after drying in the sun before and after fluorescent color selection. A is the seed with unobvious fluorescence emission of the rejected individual plant, and B is the seed with obvious fluorescence emission of the optimized individual plant.
FIG. 3 is a comparison graph of plant growth before and after bispyribac-sodium spraying at the leaf stage of 2-2.5.
FIG. 4 is an electrophoresis diagram of the fertility restorer element, the pollen lethal element and the selection marker element (fluorescent protein element, bentazon sensitive element, bispyribac-sodium resistance element) which are closely linked in the molecular marker identification vector; m1: identifying a bentazon sensitive element expression box and a seed red fluorescent protein screening element expression box, wherein P1 represents a positive control, and P2 represents a negative control; m4: identifying the bispyribac-sodium resistance element expression cassette, wherein P1 represents a positive control, and P2 represents a negative control; m6: identifying a pollen lethal gene element expression cassette, wherein P1 represents a positive control, and P2 represents a negative control; m7: and identifying the fertility restorer gene locus, which comprises an electrophoresis band corresponding to the wild type A, an electrophoresis band corresponding to the nuclear male sterile mutant a, and an electrophoresis band corresponding to a fertility restorer element (C for short, the same as the amino acid sequence coded by the A, but a plurality of nonsense SNP mutations) in the carrier.
FIG. 5 is a graph showing the result of iodine pollen staining of a preferred individual at the heading stage.
FIG. 6 shows F from the selection of inner 10B and inner 10B crossing with the intermediate maintainer line (Zhonghua 11T/Qingfeng 1B) 6 The generation is preferably an individual plant whole comparison graph.
FIG. 7 is F n-2(n≥5) To F n(n≥5) And (4) examining the planting pattern of the outcrossing robust material under the high-temperature condition by the generation.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; the reagents and plant species used in the examples are commercially available unless otherwise specified. The "rice nuclear male sterility intermediate maintainer line" and "nuclear male sterility intermediate maintainer line" in the present invention refer to a rice nuclear male sterility maintainer line to be improved.
Example 1
1. Screening of female parent
(1) A seed stage: selecting about 100 dry seeds from each single plant of the mature nuclear male sterile intermediate maintainer line by using a fluorescence microscope or a color sorter, and carrying out fluorescence color sorting on the remaining seeds for later use; eliminating single seeds with unobvious fluorescence seeds (see figure 2), sowing single fluorescent seeds and standby seeds with obvious fluorescence seeds and 1:1 fluorescence and non-fluorescent seeds ratio;
(2) seedling stage: three sets of screening elements, namely a fluorescent screening element, a bentazon sensitive element and a bispyribac-sodium resistant element, are closely linked to the carrier element T. Therefore, plants carrying the transgenic elements will exhibit sensitivity to bentazon and high resistance to herbicides such as bispyribac-sodium.
In the 3-4 leaf stage of the fluorescent seed seedlings, a commercial herbicide containing bentazon or similar chemical action components is used as a screening agent for spraying, plants carrying transgenic elements show herbicide sensitivity to cause death, and the fact that the bentazon sensitive elements work normally in the transgenic plants is verified. The method comprises the steps of spraying herbicide such as bispyribac-sodium as a screening agent in the 2-3 leaf stage of the seedling of the standby seed, killing non-transgenic plants, and showing high resistance of the herbicide such as bispyribac-sodium due to the fact that the transgenic plants carry the bispyribac-sodium resistance gene. Then, transplanting the surviving spare seed seedlings.
(3) And (3) booting: selecting excellent single plants with short plant height-medium, compact plant type, erect sword leaves, medium tillering-multiple stems and strong and/or good toughness, taking leaves, extracting DNA, carrying out vector element identification by utilizing PCR, and keeping the single plants with each element existing to continue the next test.
(4) And (3) heading stage: and (3) determining that each element exists in a single plant at the booting stage through PCR detection, continuously performing phenotype selection, selecting the single plant with high stigma exposure and lighter wrapped neck, taking mature glumous flowers, performing pollen iodine staining, and reserving the single plant with the ratio of sterile pollen to fertile pollen of 1:1 for later use.
Selecting 11T/Qingfeng 1B flowers in the nuclear male sterile intermediate maintainer line as female parent and 10B flowers in the three-line maintainer line as male parent to obtain F 1 Then, phenotype and genotype screening is carried out for a plurality of generations, and finally the rice nuclear male sterility maintainer line with stable phenotype and the genotype of aaTt is obtained. The specific operation steps of each generation are as follows:
2、F 1 screening of the plant
At F 1 Spraying bispyribac-sodium to kill non-transgenic plants in the 2-2.5 leaf stage of the generation plants (see figure 3); collecting leaves after seedling turning green, extracting DNA, identifying each carrier element by molecular marker (see figure 4), and screening to obtain F with genotype of AaTt 1 A fertile plant; during heading period to yellow ripe period of seeds, various kinds of seeds are carried out in field by naked eyesAgronomic character investigation, reserving the preferred single plant, namely reserving the exposed high stigma, lighter neck, shorter-medium plant height, compact plant type, erect sword leaf, medium-many tillers, strong stem and/or good toughness, performing pollen iodine staining on pollen (shown in figure 5), selecting the single plant with the fertility and sterility ratio of 1:1, and harvesting the single plant with normal fruit and good lodging resistance; drying the seeds in the sun, performing fluorescence color selection, and reserving F 2 A fluorescent-substituted seed,
3、F 2 screening of the plant
At F 2 Recording or marking a single plant with excellent phenotype (a single plant with exposed stigma, lighter neck, short-medium plant height, compact plant type, erect sword leaves, medium-many tillers, strong stems and/or good toughness, normal fruit and good lodging resistance) from the heading stage of a generation plant to the yellow mature period of seeds, selecting the single plant with excellent comprehensive properties and normal fruit after the seeds are mature, harvesting the self-bred seeds and taking leaves; carrying out vector element identification by using a molecular marker, and keeping the genotype as an aaTt fertile single plant seed; drying the seeds in the sun, performing fluorescence color selection, and reserving F 3 A fluorescent seed.
4、F 3 -F n(n≥5) Selection of plants with stable phenotype
According to pair F 2 The method for screening generation plants is repeated to select the phenotype, and finally the F with excellent phenotypic character and the genotype of aaTt is obtained 6 Generation stable new nuclear male sterile maintainer line (see fig. 6); meanwhile, the method needs to examine the outcrossing seed of the intermediate nuclear male sterile line with basically stable phenotype and the nuclear male sterile line with completely stable phenotype at high temperature, and comprises the following steps:
(1) According to the growth period condition, F obtained by screening n-2(n≥5) To F n(n≥5) The nuclear male sterile maintainer line and the sterile line are sown in batches, so that the heading period of all planting materials is concentrated in the months with higher temperature in Hainan, such as 5-7 months, and the daily maximum temperature is 34-38 ℃; wherein F is derived from the same individual n-2(n≥5) To F n(n≥5) The nuclear male sterile line is properly sown for several days earlier than the corresponding nuclear male sterile maintainer line, and the two lines are guaranteed to shoot at the same time or the nuclear male sterile line shoots for 1 day earlier;
(2) Come from opposite directionsF from the same individual n-2(n≥5) To F n(n≥5) The nuclear male sterile line and the maintainer line are planted according to 49 plants in 7 rows by 7 columns, the nuclear male sterile line is respectively planted in the positions of 3 rows and 4 columns, 4 rows and 3 columns, 4 rows and 4 columns, 4 rows and 5 columns and 5 rows and 4 columns, the other positions are all planted with the nuclear male sterile maintainer line from the same single plant, the planting schematic diagram is shown in figure 7, and the powder removing is carried out every day during the ear sprouting and the ear aligning;
(3) After the seeds are yellow and ripe, the fructification condition is observed by naked eyes, the seed of the nuclear male sterile line is kept as a middle-upper group, and the group with low fructification rate is eliminated.
5. Obtaining of nuclear male sterile maintainer line and nuclear male sterile line for production
Selfing the new nuclear male sterile maintainer line with stable phenotype, and performing mechanical fluorescence color selection to mass-produce fluorescent seeds (nuclear male sterile maintainer line) and non-fluorescent seeds (nuclear male sterile line), wherein the non-fluorescent seeds are used for wide testing and matching after being planted, and strong dominant hybridization combinations are screened; the fluorescent seeds are continuously used for breeding the nuclear male sterile line and the maintainer line after being planted.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (7)
1. A breeding method of a rice nuclear male sterility maintainer line with stable phenotype is characterized by comprising the following steps:
(1) Selecting rice nuclear male sterility intermediate maintainer with genotype aTt and carrier element as female parent, selecting rice material with excellent agronomic character as male parent, and hybridizing to obtain F 1 Planting seeds;
(2) To F 1 Selecting generation plants, reserving fertile plants with the genotype of AaTt, and selecting fluorescent seeds generated by single plants with excellent agronomic characters in the heading period for planting;
(3) To F 2 Selecting generation plants, reserving fertile plants with the genotype of aaTt, and selecting fluorescent seeds generated by single plants with excellent agronomic characters in the heading period for planting;
(4) Selection of F 3 To F n（n≥5） In the generation plants, fluorescent seeds generated by the single plants with excellent agronomic characters in the heading stage are a nuclear male sterile maintainer line, and non-fluorescent seeds generated by the single plants with excellent agronomic characters in the heading stage are selected as a nuclear male sterile line; wherein A represents a nuclear male sterility locus wild-type allele, and a represents a nuclear male sterility allele; t represents the presence of a carrier element, T represents the absence of a carrier element;
the male parent is a nuclear male sterile heterozygous line with the genotype of Aatt, a nuclear male sterile intermediate maintainer line with the genotype of aaTt, a three-line maintainer line with the genotype of AAtt, a two-line sterile line with the genotype of AAtt and conventional rice with the genotype of AAtt and sterile line characteristic characteristics;
the female parent is a nuclear male sterile intermediate maintainer line with sterile and fertile pollen ratio of 1:1, bentazon sensitivity, bispyribac-sodium resistance, comprehensive excellent agronomic characters and genotype of aaTt;
the carrier element in the step (1) is a fertility restorer element, a pollen lethal element and a screening marker element which are closely linked;
the screening marker element is a fluorescent protein element, a bentazon sensitive element and a bispyribac-sodium resistant element.
2. The breeding method according to claim 1, wherein the female parent in step (1) is obtained by the following method:
1) A seed stage: sowing fluorescent seeds and spare seeds of the nuclear male sterile intermediate maintainer line family with the ratio of the fluorescent seeds to the non-fluorescent seeds of 1: 1;
2) Seedling stage: spraying herbicide containing bentazone on the seedlings of the fluorescent seeds in the 3-4 leaf stage, spraying herbicide containing bentazone on the seedlings of the standby seeds in the 2-3 leaf stage, and transplanting the seedlings of the standby seeds which survive;
3) And (3) booting: selecting single plants with excellent agronomic characters, taking leaves, extracting DNA, and carrying out vector element identification by utilizing PCR;
4) And (3) heading stage: taking mature glume flowers from a single plant which is determined to exist in each element by PCR detection at the stage of booting, carrying out pollen iodine staining, and reserving the single plant with the ratio of sterile pollen to fertile pollen being 1: 1.
3. The selective breeding method according to claim 1, wherein the screening in step (2) includes screening in F 1 And (3) spraying bispyribac-sodium to kill non-transgenic plants in the seedling stage of 2-3 leaves.
4. The breeding method according to claim 1, wherein the excellent agronomic traits in steps (2) - (3) comprise: high stigma exposure, light wrapped neck, compact plant type, erect sword leaf, more tillers, strong and/or good toughness of stem, high seed setting rate, good lodging resistance, excellent rice quality, and one or more characters of sterile and fertile pollen with the ratio of 1: 1.
5. The breeding method according to any one of claims 1 to 4, further comprising the step (4) of examining the outcrossing fruit at high temperature of the nuclear male sterile line with substantially stable phenotype by:
a) According to the growth period, sowing in batches to ensure that the heading of the nuclear male sterile line is 1 day earlier than that of the nuclear male sterile maintainer line, and the daily temperature of all planting materials is 34-38 ℃;
b) The method comprises the following steps of planting 49 nuclear male sterile lines and nuclear male sterile maintainer lines of the same single plant according to 7 rows by 7 columns, respectively planting the nuclear male sterile lines at the positions of 3 rows and 4 columns, 4 rows and 3 columns, 4 rows and 4 columns, 4 rows and 5 columns and 5 rows and 4 columns, planting the nuclear male sterile maintainer lines from the same single plant at the rest positions, and performing powder removal every day during ear-picking and ear-leveling;
c) After the seeds are yellow and ripe, the fructification condition is observed, the fructification of the nuclear male sterile line is kept as an intermediate-middle population, and the population with low fructification rate is eliminated.
6. A method for mass propagation of nuclear male sterile line, characterized in that the breeding method of any one of claims 1-5 is adopted to obtain a stable phenotype rice nuclear male sterile maintainer line for selfing, and the produced non-fluorescent seeds are nuclear male sterile lines.
7. Use of the method of breeding according to any one of claims 1 to 5 or the method of claim 6 for screening a combination of dominant rice crosses.
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