CN111903501B - Method for constructing sesame recombinant inbred line - Google Patents
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Abstract
The invention relates to a method for constructing a sesame recombinant inbred line. The construction method of the sesame recombinant inbred line provided by the invention breaks through the conventional method that the method of single plant harvesting and line propagation is always adopted from F3 to F8 generations, and the same amount of capsules at the same parts are harvested and planted in a mixed manner, namely, the single plant planting is used for replacing the plant rows, and the planting area is reduced by more than 80%; in the generations from F3 to F7, by means of sparse sowing planting, seedlings are not thinned and adventitious, partial separation caused by manual selection is reduced, and labor input is saved; in the harvesting stage, capsules at the same parts of the single plants are picked to replace the traditional single-plant selection harvesting, single-plant numbering and single-plant preservation, so that the labor input is reduced, and the partial separation caused by manual selection in the harvesting process is reduced; the method is simple and easy to implement, saves labor, uses less land, needs less equipment and can well reduce the problem of group segregation caused by thinning and final singling.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a method for constructing a sesame recombinant inbred line.
Background
Sesame (Sesamum indicum L.) is one of seven major oil crops in the world, is also a traditional characteristic oil crop in China, is planted in all places, is the main product country of sesame in China, and has the total yield inferior to India. The sesame seeds are rich in unsaturated fatty acid, vitamins and microelements such as calcium, iron, zinc and the like, have good taste and quality, are beneficial to human health and are deeply loved by people. With the improvement of living standard, sesame consumption in China continuously and rapidly increases, and the sesame consumption is increased by about two times from 59 ten thousand tons in 2003 to 164 ten thousand tons in 2017, which accounts for about 30% of the total consumption of the world, and China becomes the largest sesame consuming country in the world. Therefore, the yield per unit of sesame is improved, and the method is very urgent for promoting the development of sesame production in China and solving the problem of insufficient total self-supply.
The development of genetic research on important agronomic characters of sesame and the discovery of key gene loci are important foundations for establishing a high-efficiency breeding technology and directionally cultivating excellent varieties with high yield, high quality, stress resistance, suitability for mechanization and the like. The sesame yield traits comprise individual capsule quantity, thousand kernel weight, individual plant yield and the like, the quality traits comprise oil content, protein content, sesamin content, sesamolin content, oleic acid content, linoleic acid content and the like, the stress resistance traits comprise stem withering resistance, bacterial wilt resistance, moisture resistance, drought resistance and the like, and the plant type traits suitable for mechanization comprise plant height, leaf size, leaf angle, capsule cracking resistance, lodging resistance and the like. These traits are basically quantitative traits, and constructing a suitable mapping population is the primary task for developing QTL or genetic loci.
The size of the mapped population affects the accuracy of the genetic map, and the larger the general population is, the higher the accuracy of the obtained genetic linkage map is (Guoshixing et al, 2011). However, too large a population will take a lot of unnecessary experimental time, increasing the cost of the experiment. The mapping populations used for constructing the genetic linkage map have various varieties, different populations have respective advantages and disadvantages, and the populations meeting the test requirements are selected in practical application. The classification is based on the genetic stability of the segregating population and can be divided into two categories, namely, a transient segregating population and a permanent segregating population (Jiang hong mu et al, 2008). The former includes F2, F3, BC group, etc., and the latter includes RIL, DH, NIL, NIILs, BIL, CSSL, etc. The construction of a temporally segregating population is relatively easy, but after a first generation of sexual reproduction, the genetic structure of the population is changed, and the population cannot be stored for a long time, so that the requirement of continuous research cannot be met. The genotype homozygosis between individuals in the strains in the population is permanently separated, and the genetic structure of the population is not changed after the population is propagated, so that the phenotype of the target traits of the population is inspected at multiple points for years after long-term use. The commonly used permanent population is the RIL population and the DH population. The RIL population is a segregation population generated by selfing F2 hybrid progeny for multiple generations, after 6-7 generations of selfing, the heterozygosity rate of a single locus is basically close to homozygosity, and the additive effect of the QTL can be analyzed (Livimine et al, 2000).
The high quality of the RIL population is the key to the success of gene discovery and effect analysis of the trait of interest. However, in the process of constructing the RIL population, a large area of test land is consumed each year through multi-generation strain planting, the harvesting of a single plant is time-consuming and labor-consuming, particularly in sesame, because seeds are small, in order to ensure the emergence of seedlings, the amount of the seeds is generally more than 10 times that of the seeds, then the artificial thinning is carried out for 1-2 times in the true leaf period 2-4 times after the emergence of the seedlings, and the final thinning is carried out in the true leaf period 5-6 times. In the process, due to the fact that the seedlings are different in growth vigor and the phenotypes of progeny materials are different, people generally keep plants which are vigorous in growth vigor and normal in growth, and artificially create directional selection to a certain degree. In the mature period, due to the difference of the genotype and the development of the progeny individual plant, the number of capsules of the individual plant and the number of seeds of the individual plant are greatly different, and the loss of weak plants and strains with less seeds is easily caused by conventional individual plant selfing and individual plant harvest and storage, so that the phenotype segregation of the constructed RIL group is serious, and the accurate positioning of the target character gene locus is difficult.
Aiming at the problems, the construction method of the sesame recombinant inbred line which is simple, convenient and labor-saving and can effectively reduce partial segregation is provided, the application of the method is favorable for improving the construction efficiency and quality of sesame RIL groups, and the method plays an important role in developing sesame target character QTL or gene locus discovery.
Disclosure of Invention
One of the technical problems to be solved by the present invention is to provide a method for constructing a sesame recombinant inbred line, which can effectively solve the problem of segregation of group traits caused by manual thinning and final singling in the process of constructing the recombinant inbred line by a conventional method, and has the advantages of less manpower, test land and equipment, and simplicity and convenience.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the construction method of the sesame recombinant inbred line comprises the following steps:
(1) selecting two homozygous parent materials with obviously different target properties, planting in a net shed in an isolated manner, selecting single male parent plants and single female parent plants in a flowering period to perform plant-to-plant hybridization, harvesting F0 generation hybrid seeds, male parents and female parents after normal maturity, and marking;
(2) planting F1 generation and male parent and female parent plant rows in the 2 nd growing season, thinning and fixing seedlings conventionally, and harvesting normally mature F1 generation plants in the mature period after determining that the F1 generation plants are homozygous hybrid seeds through investigation; respectively harvesting a certain amount of male parents and female parents according to the requirements and preserving for later use;
(3) in 3 rd growing season, planting F2 generation in sparse sowing, not thinning, fixing seedlings, and performing other normal field management, when the plants are normally mature, picking up the same amount of normal capsules from the same parts of each plant, mixing, airing and threshing;
(4) planting harvest groups F3 to F6 and other generations in the following growing season according to the method described in (3);
(5) planting mixed seeds in the F7 generation, and harvesting and numbering the single plants in the mature period;
(6) in the F8 generation, planting the harvested F7 and male parent and female parent according to the strains, normally thinning and setting seedlings, respectively and mixedly collecting the male parent, female parent and seeds of each F8 strain according to the strains in the mature period, numbering and storing for later use, thereby completing the construction of the recombinant inbred line.
According to the scheme, the step (2) is as follows: and (4) respectively inspecting and determining whether the F1 generation plants are homozygous hybrid seeds at the seedling stage and the flowering stage, and otherwise, reconfiguring the hybrid combination.
According to the scheme, at least 3 normally mature F1 generation plants are harvested in the step (2), and the seeds are mixed, aired and threshed, wherein the harvested seed amount is not less than 10 g.
According to the scheme, in the F2 generation of sparse sowing planting in the step (3), the distance between the sowed seeds is more than 5 cm.
According to the scheme, the number of the normal capsules extracted in the step (3) is 1-5.
The invention has the advantages that:
the construction method of the sesame recombinant inbred line provided by the invention breaks through the conventional method that the method of single plant harvesting and line propagation is always adopted from F3 to F8 generations, and the same amount of capsules at the same parts are harvested and planted in a mixed manner, namely, the single plant planting is used for replacing the plant rows, and the planting area is reduced by more than 80%;
in the method, in the generations from F3 to F7, by means of sparse sowing planting, seedlings are not thinned and adventitious, partial separation caused by manual selection is reduced, and the labor input is saved;
according to the method, capsules at the same parts of the single plants are picked in the harvesting stage, and the traditional single-plant selection harvesting, single-plant numbering and single-plant preservation are replaced, so that the labor input is reduced, and the partial separation caused by manual selection in the harvesting process is reduced.
In conclusion, the invention provides a simple and labor-saving construction method of the sesame recombinant inbred line, which can effectively reduce partial segregation, greatly improve the construction efficiency and quality and save the cost.
Drawings
FIG. 1 shows the strain height distribution of a sesame strain height recombinant inbred line population F8. The diagram shows that the population constructed by the method provided by the invention is in normal distribution and has no obvious segregation.
The following examples are intended to illustrate the invention without limiting its scope of application.
Example (b): sesame plant height Recombinant Inbred Line (RIL) group construction
(1) In summer of 2013, in the Wuchang experiment base of the institute of oil crops, Chinese academy of agricultural sciences, high-stalk sesame material AG150 (with a plant height of 1.8m) is selected as a male parent and low-stalk material AG151 (with a plant height of 80cm) is selected as a female parent, the high-stalk sesame material is planted in a net shed isolation mode, two male parents (AG150 r and AG150 ②) and two female parents (AG151 r and AG152 (II)) are respectively selected in a flowering period to carry out plant-to-plant hybridization, the male parent, the female parent and F0 generation hybrid seeds are harvested after normal maturity, and the listing marks are ZW105 r and ZW105 er.
(2) In 2013 winter, F1 generations of ZW105 & lt- & gt, male parent AG150 & lt- & gt and AG150 & lt- & gt, female parent AG151 & lt- & gt and AG152 & lt- & gt are planted in Hainan Mitsui by using the harvested F0 generations of hybrid seeds, conventional thinning and final singling are carried out, F1 generations and parent characters are inspected and compared in seedling stage and flowering stage, the obtained ZW105 & lt- & gt hybrid combination is determined to be a homozygous true hybrid, and the separated ZW105 & lt- & gt is abandoned; 5 normally mature ZW105 plants are harvested in a mixed mode in the mature period, and the seeds are aired and threshed, wherein the harvested seed amount is 68.5 g; respectively mixing and harvesting 5 strains of the corresponding male parent AG 150I and female parent AG 151I, and storing for later use;
(3) f2 generations are planted in summer of 2014 in Wuhan, ZW105 (the first one) seeds are sown at a low density, and the distance between the seeds is more than 5 cm; thinning and adventitious seeding are not carried out after seedling emergence, other normal field management is carried out, when plants are normally mature, 2 normal capsules are picked from the middle part of each plant, and the plants are mixed, aired and threshed, and mixed according to the number AGF 2;
(4) in the subsequent growing season, planting and harvesting populations F3 to F6 and other generations are numbered AGF3 mixed, AGF4 mixed, AGF5 mixed and AGF6 mixed according to the method described in (3);
(5) in the F7 generation, mixed AGF6 seeds are planted in a sparse sowing mode, and single plants are harvested in the mature period and are numbered A001, A002 and A003 … … A686;
(6) in the F8 generation, planting the harvested F7 single plants A001, A002, A003 … … A686 and the male parent and the female parent according to plant lines, normally thinning and setting seedlings, respectively mixing and harvesting the male parent, the female parent and 686F 8 plant line seeds, numbered AGP1, AGP2, AGH001, AGH002 and AGH003 … … AGH686 according to the plant lines in the maturation period, properly preserving for later use, and finishing the construction of a recombinant inbred line;
(7) planting AGH001, AGH002 and AGH003 … … AGH686, planting 3 rows in each plant line, normally thinning and final singling, measuring the plant height in the final flowering phase, investigating the variation distribution of the plant height (figure 1), and analyzing results show that the plant height variation of the plant group is in a continuous quantitative character, conforms to the normal distribution characteristic, has no obvious segregation, and shows that the plant height recombinant inbred line group is successfully constructed.
Claims (5)
1. A construction method of a sesame recombinant inbred line is characterized by comprising the following steps: the method comprises the following steps:
(1) selecting two homozygous parent materials with obviously different target properties, planting in a net shed in an isolated manner, selecting single male parent plants and single female parent plants in a flowering period to perform plant-to-plant hybridization, and harvesting F0 generation hybrid seeds, male parents and female parents after normal maturity;
(2) planting F1 generation and male parent and female parent plant rows in the 2 nd growing season, thinning and fixing seedlings conventionally, and harvesting normally mature F1 generation plants in the mature period after the F1 generation plants are determined to be true hybrid seeds through investigation; respectively harvesting corresponding male parent and female parent according to the requirement;
(3) in the 3 rd growing season, F2 generation is sparsely sown and planted, seedlings are not thinned and fixed, other normal field management is carried out, when plants are normally mature, the same amount of normal capsules are picked from the same part of each plant, and the normal capsules are mixed, aired, threshed and harvested;
(4) planting the harvest population F3 to F6 generation in the following growing season according to the method described in (3);
(5) planting mixed seeds in the F7 generation, and harvesting and numbering the single plants in the mature period;
(6) in the F8 generation, the harvested F7 and the single plants of the male parent and the female parent are planted according to the strains, normal thinning and final singling are carried out, seeds of the male parent, the female parent and each F8 strain are mixed according to the strains in the mature period and are numbered, and the construction of the recombinant inbred line is completed.
2. The method of claim 1, wherein: the step (2) is as follows: and (4) respectively inspecting and determining whether the F1 generation plants are true hybrids in the seedling stage and the flowering stage, and otherwise, reconfiguring the hybrid combination.
3. The method of claim 1, wherein: and (3) harvesting not less than 3 normally mature F1 generation plants in the step (2), mixing, airing and threshing, wherein the amount of harvested seeds is not less than 10 g.
4. The method of claim 1, wherein: in the F2 generation of sparse sowing planting in the step (3), the distance between the sowed seeds is more than 5 cm.
5. The method of claim 1, wherein: the number of the normal capsules extracted in the step (3) is 1-5.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525919A (en) * | 2013-09-27 | 2014-01-22 | 中国农业科学院油料作物研究所 | Molecular marker tightly linked with main effective genetic locus embodying sesame dampness resistance and application thereof |
| CN107593314A (en) * | 2017-08-29 | 2018-01-19 | 江西省农业科学院蔬菜花卉研究所 | A kind of construction method of balsam pear More female lines recombinant inbred lines |
| CN109601367A (en) * | 2018-12-06 | 2019-04-12 | 南京农业大学 | The breeding method of downy mildew resistance cucumber interspecific hybrid new varieties and application |
| CN110089355A (en) * | 2019-04-26 | 2019-08-06 | 中国农业科学院油料作物研究所 | A kind of sesame mitigation high quality type of seeding |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525919A (en) * | 2013-09-27 | 2014-01-22 | 中国农业科学院油料作物研究所 | Molecular marker tightly linked with main effective genetic locus embodying sesame dampness resistance and application thereof |
| CN107593314A (en) * | 2017-08-29 | 2018-01-19 | 江西省农业科学院蔬菜花卉研究所 | A kind of construction method of balsam pear More female lines recombinant inbred lines |
| CN109601367A (en) * | 2018-12-06 | 2019-04-12 | 南京农业大学 | The breeding method of downy mildew resistance cucumber interspecific hybrid new varieties and application |
| CN110089355A (en) * | 2019-04-26 | 2019-08-06 | 中国农业科学院油料作物研究所 | A kind of sesame mitigation high quality type of seeding |
Non-Patent Citations (1)
| Title |
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| 亚麻SNP遗传图谱的构建及品质相关性状的QTL定位研究;高凤云;《中国博士学位论文全文数据库 农业科技辑》;20180630;第20页 * |
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