CN108935068B - Method for improving fertility temperature sensitivity of brassica napus pol CMS sterile line - Google Patents
Method for improving fertility temperature sensitivity of brassica napus pol CMS sterile line Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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Abstract
The invention discloses a method for improving fertility temperature sensitivity of a brassica napus pol CMS sterile line, which comprises the following steps: respectively hybridizing a seed material to be transferred serving as a male parent and an existing pol CMS sterile line A and a maintainer line B serving as female parents to obtain A-F1 and B-F1; selfing B-F1 to obtain B-F2, and hybridizing the B-F1 serving as a male parent with A-F1 to obtain A-BC 1; selecting A-BC1 with less micropowder as a female parent to hybridize with B-F2 to obtain A-BC2, and selfing and reserving seeds of the B-F2 to obtain B-F3; and carrying out positive and negative crossing on the A-BC2 with less micropowder and the B-F3 to obtain a reverse crossing generation B-wF1 and an orthogonal generation A-BC3, B-wF1, continuously selfing for two generations, and synchronously backcrossing an A line for two generations. The method of the invention accelerates the stabilization process of the temperature insensitive character in the pol CMS maintainer line transferring process, thereby effectively improving the sterile stability and the sterile thoroughness of the pol CMS sterile line.
Description
Technical Field
The invention belongs to the technical field of agricultural planting, cultivation and breeding, and particularly relates to a method for improving fertility temperature sensitivity of a brassica napus pol CMS sterile line.
Background
The cabbage type rape polima cytoplasmic male sterile line (pol CMS) is a sterile system discovered by Huazhong agriculture great Fourier courtyard in 1972, and three-line matching (sterile line A, maintainer line B and restorer line R) is realized by the Hunan province crop research institute in 1976, so that the cabbage type rape CMS is internationally recognized as the first cabbage type rape CMS type with practical value. In recent 20 years, the system has cultivated over 60 percent of hybrid rape varieties in China, and is one of indispensable key technologies for utilizing the heterosis of rape in China. However, the sterile line of the system has the defects of sensitive flowering phase to temperature and unstable fertility, and brings great risk to hybrid seed production, so that the key point for reducing the risk of hybrid seed production is to obtain the sterile line which is insensitive to temperature change.
Through years of research, it is found that the sensitivity of different types of pol CMS to temperature mainly depends on the interaction between the nuclear temperature sensitive gene and the sterile gene. Due to the complexity of the generation reasons of the trace pollen of the pol CMS sterile line, the transformation efficiency of the sterile line with relatively insensitive temperature is not high all the time, and the popularization and application of the sterile line on a larger and higher level are limited. Therefore, improving the fertility temperature sensitivity of the brassica napus pol CMS sterile line to improve the production and application defects of the sterile line of the system is the direction of continuous efforts of technical personnel in the research and application fields.
Disclosure of Invention
The technical problem to be solved by the present invention is to overcome the technical defects mentioned in the background art, and to provide a method for improving fertility temperature sensitivity of a brassica napus pol CMS sterile line, which can effectively improve the sterility stability of the sterile line.
In order to solve the technical problems, the technical scheme provided by the invention is to provide a method for improving fertility temperature sensitivity of a brassica napus pol CMS sterile line, which comprises the following steps:
(1) respectively hybridizing a seed material to be transferred serving as a male parent and an existing pol CMS sterile line A and a corresponding maintainer line B serving as female parents to obtain hybrid progeny A-F1 population (semi-sterile population) and B-F1 population seeds;
(2) planting the seeds of the A-F1 population and the B-F1 population obtained in the step (1) in different regions, selecting individual plants of the B-F1 population (selecting individual plants with excellent comprehensive properties) at the flowering phase, bagging, isolating and selfing to obtain a B-F2 population, and hybridizing the bagged individual plants of the B-F1 population serving as male parents with sterile individual plants of the A-F1 population to obtain the seeds of a hybrid progeny A-BC1 population (separating a small amount of blunt-sense sterile individual plants);
(3) planting the seeds of the B-F2 and A-BC1 populations obtained in the step (2) in a subarea, observing fertility performance of the A-BC1 population in a flowering phase, selecting and reserving the A-BC1 population with less micropowder generation, selecting a single plant with less micropowder as a female parent in the population and hybridizing the single plant with less micropowder in the population with the single plant in the B-F2 population to obtain the A-BC2 seeds (less insensitive sterile separation); meanwhile, selfing and reserving seeds of the hybridized B-F2 single plant (selecting the single plant with excellent comprehensive properties) to obtain seeds of a B-F3 group;
(4) planting the seeds of the B-F3 and A-BC2 populations obtained in the step (3) in a subarea, observing fertility performance of A-BC2 in a flowering phase, selecting and reserving the A-BC2 population with less micropowder generation, selecting a single plant with less micropowder generation and performing positive and negative crossing with a corresponding single plant of the B-F3 population, and respectively obtaining seeds of a B-wF1 (temperature insensitive fixation) population of a reverse crossing progeny and seeds of an A-BC3 population of an orthogonal progeny (obviously improved sterility);
(5) and (3) planting the seeds of the B-wF1 and A-BC3 populations obtained in the step (4) in a subarea, backcrossing the B-wF2 obtained after the B-wF1 is selfed with the A-BC3 to obtain the A-BC4 (to obtain a temperature-insensitive sterile line), further selfing the B-wF2 to obtain the B-wF3 obtained after the B-wF2 is selfed with the A-BC4 to obtain the A-BC5, and obtaining the stable temperature-insensitive pol CMS sterile line A and the corresponding maintainer line B thereof, so that the improvement of the fertility temperature sensitivity of the brassica napus pol CMS sterile line is completed.
In the method, the seed material to be transferred selected in the step (4) is a new germplasm with obviously excellent properties, and the invention is preferably Zhejiang oil 50 with the oil content of 48-49%.
Preferably, the existing sterile line selected in the step (1) is a sterile line with strong general combining ability and stable sterile character, the existing pol CMS sterile line A preferred in the invention is 20A, and the corresponding maintainer line B is 20B (10 examined varieties are bred as parents).
Preferably, in the reciprocal crossing of the step (4), the reciprocal crossing is to pollinate the emasculated individual of the B-F3 population to obtain the B-wF1 population by taking a trace amount of pollen of the sterile individual of the A-BC2 population from 11 to 12 am on a cloudy day.
Preferably, in the step (4), the reciprocal crossing of B-F3 and A-BC2 is performed when the basic biological characters of the two populations are similar, and the step (3) is repeated if the basic biological characters of the two populations are obviously different, until the basic biological characters of the two populations are similar, the step (4) is not performed.
Preferably, the two populations have similar basic biological characteristics, which means that the two populations have similar oil content, plant height and branch number. More preferably, the oil content of the two groups is more than 45%, the plant height is less than 170 cm, and the branch number is more than 6.
Preferably, in the step (5), the fertility of the temperature-insensitive pol CMS sterile line is less affected by a change in light temperature, the occurrence of micropowder is reduced, and the ratio of stamen to pistil length is less than 1/3.
The invention is based on the principle that: the temperature insensitive is quantitative character which can only be shown on sterile plants, and the length ratio of stamens to pistils on the sterile plants is more than 1:3, more than 1:2 is half sterile, and less than 1:3 is considered as less micro powder. The temperature insensitive character of the sterile plant is better (the length ratio of stamens to pistils is less than 1/3), which indicates that the temperature insensitive gene exists in the nuclear genome, and the remaining trace pollen is used for transferring the maintainer line, so that the target gene can be transferred into the maintainer line to the maximum extent, and the target property of the maintainer line is improved.
According to the technical scheme, the maintainer line is used as a male parent to be orthogonal with the sterile line, and the key technology of reverse crossing and transferring of the maintainer line by transferring trace pollen of the sterile line is adopted, so that the stable process of the temperature insensitive character in the transferring process of the pol CMS maintainer line is accelerated, the sterile stability and the sterile thoroughness of the pol CMS sterile line are effectively improved, and the method has reference value in transferring other CMS sterile lines sensitive to temperature.
Compared with the prior art, the invention has the beneficial effects that:
1. the method can change the problem that the genetic conservation of the maintainer line is difficult to improve in the transformation process of the pol CMS sterile line, accelerates the stability of fertility insensitive genetic components and the sterility thoroughness by the positive and negative crossing of the single plant in the sterile population and the single plant in the maintainer population, thereby improving the fertility maintaining effect of the maintainer line, further realizes the improvement of the fertility temperature sensitivity of the cabbage type rape pol CMS sterile line, can transform the visible sterile phenotypic characters on the sterile population to the maintainer line in a reverse way on the basis of the orthogonal transformation and wide screening of the maintainer line, converts the internal quantitative characters into the visible phenotypic characters for selection, and greatly improves the transformation goal and transformation efficiency.
2. The sterile line cultivated by the method of the invention has high general matching force, the prepared hybrid combination has obvious yield increase potential, a plurality of cabbage type rape pol CMS sterile lines (such as H20A) are cultivated according to the method at present, and the newly cultivated sterile line has stable fertility, slow hypo-induction reaction and thorough sterility.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic representation of a breeding scheme of an embodiment.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various starting materials for use in the present invention are either commercially available or can be prepared by existing methods.
Example (b):
the method for improving fertility temperature sensitivity of the brassica napus pol CMS sterile line disclosed by the invention is shown in figure 1 and comprises the following steps of:
(1) in 2008, Zhe oil 50, a conventional variety bred by Zhejiang agricultural institute, Hunan province, utilizes as a male parent, the oil content of which is 48-49%, and pol CMS sterile line 20A and maintainer line 20B bred by the unit are respectively used as female parents to be hybridized to obtain F1 filial generations: A-F1, B-F1;
(2) in 2009, the seeds of the A-F1 and B-F1 populations obtained in step (1) are planted in different regions, and it can be seen that A-F1 is a semi-sterile population, and the length ratio of stamens to pistils is about 1/2; selecting individual plants of a B-F1 group with excellent comprehensive properties at the flowering phase, bagging, isolating and selfing to obtain a B-F2 group, and hybridizing the bagged B-F1 group individual plants serving as male parents with sterile individual plants of an A-F1 group to obtain a small amount of seeds of a hybrid progeny A-BC1 group separated from the insensitive sterile individual plants;
(3) planting the seeds of the B-F2 and A-BC1 populations obtained in the step (2) in a subarea, observing fertility performance of A-BC1 in a flowering period, wherein about 90% of micropowder is more, but about 10% of individuals are insensitive to temperature (the micropowder is less), fertility is less influenced by light and temperature, the micropowder is particularly less, the length ratio of stamens to pistils is less than 1/3, selecting the individuals with less micropowder as female parents to hybridize with the B-F2 population individuals with excellent comprehensive properties such as quality, resistance and biological properties as male parents to obtain a small amount of sterile hybrid progeny A-BC2 seeds which are insensitive to sterility and separated, and simultaneously selfing the hybridized male parents B-F2 individuals to obtain the seeds of a progeny B-F3 population;
(4) planting the seed groups of the B-F3 and A-BC2 groups obtained in the step (3) in a subarea, observing the fertility performance of the A-BC2 group in the flowering period, wherein the field appearance characters (except flower organs) of the two groups are similar (the oil content, the plant height and the branch number are similar, and the basic biological characters are similar), the micro powder performance of the group single plants is obviously different, but only about 10 percent of the single plants are temperature insensitive, and selecting the temperature insensitive single plants with less micro powder to be subjected to positive and negative hybridization with the B-F3 group single plants, so as to respectively obtain orthogonal progeny A-BC3 with obviously improved sterility and reverse-crossing progeny B-wF1 seeds with fixed temperature insensitive; wherein, the micro powder reverse crossing is to pollinate the emasculated individual plant of the B-F3 population by taking the residual pollen of the sterile individual plant of the A-BC2 population from 11 to 12 am on the cloudy day to obtain a B-wF1 population;
the positive and negative crossing of B-F3 and A-BC2 is carried out when the basic physical properties of the two populations are similar, if the basic physical properties of the two populations are obviously different, the step (3) is repeated until the basic physical properties of the two populations are similar, the step (4) is carried out, the oil content of the two populations is more than 45 percent, the plant height is less than 170 cm, and the branch number is more than 6;
(5) planting seeds of the B-wF1 and A-BC3 populations obtained in the step (4) in a district, and then continuously selfing B-wF1 and backcrossing the seeds with A-BC3 for 2 generations, namely, carrying out backcrossing on B-wF2 obtained after selfing B-wF1 to obtain A-BC4 by using A-BC3, and carrying out continuous selfing on B-wF2 to obtain B-wF3 and backcrossing on A-BC4 to obtain A-BC5 by using B-wF 3; the field apparent characters (except flower organs) of the two groups are basically consistent (the oil content, the plant height and the branch number are similar, and can be regarded as similar basic biological characters), the stable temperature insensitive pol CMS sterile line A and the corresponding maintainer line B are obtained and are named as H20A and H20B, and the improvement of the fertility temperature sensitivity of the brassica napus pol CMS sterile line is completed.
After the new B line (maintenance line) is selected for comprehensive characters such as quality, resistance, biological characters and the like, the oil content is more than 45%, the height of the plant is less than 170 cm, and the number of branches is more than 6, so that the sterile stability of the obtained new A line (sterile line) is remarkably improved, the single plant of the temperature insensitive sterile line reaches more than 70%, and the expected improvement effect is met.
Claims (2)
1. A method for improving fertility temperature sensitivity of a cabbage type rape pol CMS sterile line comprises the following steps:
(1) respectively hybridizing a seed material to be bred serving as a male parent and an existing pol CMS sterile line A and a corresponding maintainer line B serving as female parents to obtain hybrid progeny A-F1 population and B-F1 population seeds; the seed material to be transferred is Zhe oil 50, the pol CMS sterile line A is 20A, and the corresponding maintainer line B is 20B;
(2) planting the seeds of the A-F1 population and the B-F1 population obtained in the step (1) in different regions, selecting a B-F1 population single plant in a flowering phase, bagging, isolating and selfing to obtain a B-F2 population, and hybridizing the bagged B-F1 population single plant serving as a male parent with the A-F1 population sterile single plant to obtain a hybrid progeny A-BC1 population seed;
(3) planting the seeds of the B-F2 and A-BC1 populations obtained in the step (2) in a subarea, observing fertility performance of the A-BC1 population in a flowering phase, selecting and reserving the A-BC1 population with less micropowder generation, selecting a single plant with less micropowder as a female parent in the population and hybridizing the single plant with less micropowder in the B-F2 population to obtain an A-BC2 seed; meanwhile, selfing and reserving seeds of the hybridized B-F2 single plant to obtain seeds of a B-F3 group;
(4) planting the seeds of the B-F3 and A-BC2 populations obtained in the step (3) in a subarea, observing fertility performance of A-BC2 in a flowering phase, selecting and reserving the A-BC2 population with less micropowder generation, selecting a single plant with less micropowder generation and a corresponding B-F3 population single plant for positive and negative crossing, and respectively obtaining seeds of a B-wF1 population of a reverse crossing progeny and seeds of an A-BC3 population of an orthogonal progeny; in the positive and negative crossing, the reverse crossing is to pollinate the emasculated individual plant of the B-F3 population by taking trace pollen of the sterile individual plant of the A-BC2 population from 11 to 12 am on the cloudy day to obtain a B-wF1 population; the positive and negative crossing of B-F3 and A-BC2 is carried out when the basic biological characters of the two populations are similar, if the basic biological characters of the two populations have obvious difference, the step (3) is repeated, and the step (4) is not carried out until the basic biological characters of the two populations are similar;
(5) and (3) planting seeds of the B-wF1 and A-BC3 populations obtained in the step (4) in a subarea, backcrossing B-wF2 obtained after B-wF1 selfing with A-BC3 to obtain A-BC4, further selfing B-wF2 to obtain B-wF3 obtained after B-wF2 selfing, and backcrossing A-BC4 to obtain A-BC5 to obtain a stable temperature insensitive pol CMS sterile line A and a corresponding maintainer line B thereof, thus finishing the improvement of the fertility temperature sensitivity of the brassica napus pol CMS sterile line.
2. The method according to claim 1, wherein in the step (5), the sterility of the temperature-insensitive pol CMS sterile line is less affected by temperature changes, the occurrence of micropowder is less, and the ratio of stamen to pistil length is < 1/3.
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