CN103749286B - A kind of construction method covering wild rice full-length genome hybrid fragments introgressive line - Google Patents
A kind of construction method covering wild rice full-length genome hybrid fragments introgressive line Download PDFInfo
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- CN103749286B CN103749286B CN201410009724.2A CN201410009724A CN103749286B CN 103749286 B CN103749286 B CN 103749286B CN 201410009724 A CN201410009724 A CN 201410009724A CN 103749286 B CN103749286 B CN 103749286B
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Abstract
The present invention relates to a kind of construction method covering wild rice full-length genome hybrid fragments introgressive line, belong to biological technical field, by continuous backcross and selfing, and by means of molecular marker assisted selection, and build hybrid fragments introgressive line colony, to reach the speed that improves gene clone and to reduce experimental cost object, be that the study mechanism of rice heterosis provides ideal basic material for carrying out.Wild rice contains abundant beneficial gene source, comprise the tolerance gene etc. of various high-quality, high yield, damage by disease and insect resistance and abiotic stress, hybrid fragments introgressive line informative population, is conducive to identifying these proterties equipotential or new gene, is conducive to the hereditary basis enriching cultivated rice.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of construction method covering wild rice full-length genome hybrid fragments introgressive line.
Background technology
Paddy rice is one of most important cereal crops in the world.Along with cultivating the cultivation and popularization of improving the breed in a large number, rice yield has had large increase.But be subject to the impact of various biology and abiotic stress due to paddy rice, and it is comparatively single to promote hereditary basis of improving the breed at present.Therefore, expand the available genetic resources of cultivated rice, increase yield potentiality and the adverse circumstance defensive ability/resistance ability of cultivated rice, constantly improve the problem that rice yield has become current urgent solution.Wild rice is an important beneficial gene source, comprises the tolerance gene etc. of various high-quality, high yield, damage by disease and insect resistance and abiotic stress.But wild rice economical character is poor, genetic background is quite complicated, the unfavorable gene frequency of occurrences is higher, interaction of genes increases, many beneficial genes cover by its background, and to be difficult to its precise Identification by traditional phenotypic evaluation method.Obviously, by building the chromosome segment introgressive line colony be donor cultivated rice with wild rice being acceptor, the impact of genetic background can be eliminated, create condition for excavating further and locating favorable genes in wild rice.
Full-length genome hybrid fragments introgressive line colony utilizes continuous backcross and selfing that donor chromatin fragment is imported to receptor parent, simultaneously by means of molecular marker assisted selection, and obtains the heterozygosis introgressive line colony covering donor parents full-length genome.
Traditional introgressive line informative population utilizes continuous backcross and selfing to import in receptor parent by donor chromatin fragment, major part is all in self progeny, only select to isozygoty the individual plant of fragment containing genes of interest, but abandon the hybrid fragments individual plant containing genes of interest.And when the later stage accurately locates certain gene, rebuild the heterozygosis strain with receptor parent again, obviously have impact on the process of experiment and add experimental cost.
Summary of the invention
The object of the present invention is to provide a kind of construction method covering wild rice full-length genome hybrid fragments introgressive line, by continuous backcross and selfing, and by means of molecular marker assisted selection, and build hybrid fragments introgressive line colony, to reach the speed that improves gene clone and to reduce experimental cost object, be that the study mechanism of rice heterosis provides ideal basic material for carrying out.
For achieving the above object, the present invention adopts following technical scheme:
Cover a construction method for wild rice full-length genome hybrid fragments introgressive line, it mainly comprises Juvenile stage, hybridizes, backcrosses, target label detection, selfing, target label detect; Concrete steps are as follows:
(1) Juvenile stage: donor parents is wild rice, receptor parent requires there is notable difference with wild rice genetic background;
(2) parents: receptor parent is maternal, and donor parents is male parent, hybridization individual plant general random 3-5 strain;
(3) backcross: from F
1in generation, starts, and Stochastic choice 20-25 strain and receptor parent backcross, and obtain BC
1f
1; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain 55-65 BC
2f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain the BC of 170-190
3f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and finally obtain 530-550 BC
4f
1strain; Then when running into the hybrid fragments of disappearance, be at BC secondly,
2f
1for reserve part strain, the later stage only screens with disappearance target label, is backcrossed by the individual plant containing disappearance target gene again;
(4) target label detect: first with 23-25 to the good primer of polymorphism, average every bar chromosome 2 pairs of primers, be distributed in chromosomal long-armed with on galianconism respectively, all individualities are analyzed, the individual plant of screening containing less than 1 or 1 hybrid fragments, then carries out full-length genome genotyping to the individual plant retained;
(5) selfing: after full-length genome genotyping is carried out to individual plant, choose the individual plant selfing having less than 2 hybrid fragments, and carry out bagging, then sowing;
(6) target label detects: selected single-strain planting, containing 1 hybrid fragments each strain plantation 50-300 strain, and each strain plantation 100-400 strain containing 2 hybrid fragments;
(7) hybrid fragments introgressive line individual plant screening: with target label, detection is carried out to plantation strain individual plant and analyze, select containing target label heterozygosis individual plant; Also retain containing the target label individual plant that isozygotys simultaneously.
Described receptor parent is long-grained nonglutinous rice or japonica rice.
Selected individual plant in described step (6) is BC
3f
2or BC
4f
2the seed in generation, will amplify colony as far as possible, could obtain the individual plant of target label; Heterozygosis individual plant in step (7) is BC
3f
3or BC
4f
3the seed in generation, does not need to plant again, because individual plant is heterozygosis individual plant.
The invention has the advantages that:
(1) build hybrid fragments introgressive line colony, the speed of gene location clone can be significantly improved and reduce experimental cost.
(2) each strain of hybrid fragments introgressive line colony is only the heterozygosis in a certain section of region, and other region is and isozygotys, and is conducive to studying the aspect such as mutual work, heterotic mechanism between wild rice and cultivated rice allelomorph.
(3) wild rice contains abundant beneficial gene source, comprise the tolerance gene etc. of various high-quality, high yield, damage by disease and insect resistance and abiotic stress, hybrid fragments introgressive line informative population, is conducive to identifying these proterties equipotential or new gene, is conducive to the hereditary basis enriching cultivated rice.
Embodiment
embodiment 1
Take wild rice as donor parents, with long-grained nonglutinous rice material (as 9311, bright extensive 63 etc.) for receptor parent, by continuous backcross and selfing, and by means of Molecular Marker Assisted Selection Technology, can obtain with long-grained nonglutinous rice is that background covers wild rice full-length genome hybrid fragments introgressive line colony.Concrete steps are:
(1) Juvenile stage: donor parents is wild rice, receptor parent is 9311, and receptor parent General Requirements and wild rice genetic background have notable difference.
(2) parents: General Requirements receptor parent is maternal, and donor parents is paternal hybrid, the strain of hybridization individual plant general random 3.
(3) backcross: from F
1in generation, starts, and Stochastic choice 20 strain and receptor parent backcross, and obtain BC
1f
1; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain 60 BC
2f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain the BC of 180
3f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and finally obtain 540 BC
4f
1strain; Next, then when running into a small amount of hybrid fragments lacked, be generally at BC
2f
1for reserve part strain, the later stage only screens with disappearance target label, is backcrossed by the individual plant containing disappearance target gene again.
(4) target label detects: first use 24 pairs of good primers of polymorphism, average every bar chromosome 2 pairs of primers, be distributed in chromosomal long-armed with on galianconism respectively, all individualities are analyzed, the individual plant of screening containing less than 1 or 1 hybrid fragments, then carries out full-length genome genotyping to the individual plant retained.
(5) selfing: after full-length genome genotyping is carried out to individual plant, choose the individual plant selfing having less than 2 hybrid fragments, and carry out bagging, then sowing.
(6) target label detects: selected single-strain planting is (because selected individual plant is BC
3f
2or BC
4f
2the seed in generation, will amplify colony as far as possible, could obtain the individual plant of target label), plant 100 strains containing 1 each strain of hybrid fragments, each strain containing 2 hybrid fragments plants 150 strains.
(7) hybrid fragments introgressive line individual plant screening: carry out detection with target label to plantation strain individual plant and analyze, (these individual plants are BC to select to contain target label heterozygosis individual plant
3f
3or BC
4f
3the seed in generation, does not need to plant again, because individual plant is heterozygosis individual plant); Also retain containing the target label individual plant that isozygotys simultaneously.
embodiment 2
Take wild rice as donor parents, with japonica rice material (as fine in Japan, 02428 etc.) for receptor parent, by continuous backcross and selfing, and by means of Molecular Marker Assisted Selection Technology, can obtain with japonica rice is that background covers wild rice full-length genome hybrid fragments introgressive line colony.Concrete steps are:
(1) Juvenile stage: donor parents is wild rice, receptor parent is that Japan is fine, and receptor parent General Requirements and wild rice genetic background have notable difference.
(2) parents: General Requirements receptor parent is maternal, and donor parents is paternal hybrid, the strain of hybridization individual plant general random 5.
(3) backcross: from F
1in generation, starts, and backcrosses, obtain BC1F1 about Stochastic choice 25 strain with receptor parent; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain 65 BC
2f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain the BC of 190
3f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and finally obtain 550 BC
4f
1strain; Next, then when running into a small amount of hybrid fragments lacked, be generally at BC
2f
1for reserve part strain, the later stage only screens with disappearance target label, is backcrossed by the individual plant containing disappearance target gene again.
(4) target label detects: first use 24 pairs of good primers of polymorphism, average every bar chromosome 2 pairs of primers, be distributed in chromosomal long-armed with on galianconism respectively, all individualities are analyzed, the individual plant of screening containing less than 1 or 1 hybrid fragments, then carries out full-length genome genotyping to the individual plant retained.
(5) selfing: after full-length genome genotyping is carried out to individual plant, choose the individual plant selfing having less than 2 hybrid fragments, and carry out bagging, then sowing.
(6) target label detects: selected single-strain planting is (because selected individual plant is BC
3f
2or BC
4f
2the seed in generation, will amplify colony as far as possible, could obtain the individual plant of target label), plant 150 strains containing 1 each strain of hybrid fragments, each strain containing 2 hybrid fragments plants 200 strains.
(7) hybrid fragments introgressive line individual plant screening: carry out detection with target label to plantation strain individual plant and analyze, (these individual plants are BC to select to contain target label heterozygosis individual plant
3f
3or BC
4f
3the seed in generation, does not need to plant again, because individual plant is heterozygosis individual plant); Also retain containing the target label individual plant that isozygotys simultaneously.
embodiment 3
Take wild rice as donor parents, with the material of particular phenotype proterties (as black rice, purple rice, red rice etc.) for receptor parent, by continuous backcross and selfing, and by means of Molecular Marker Assisted Selection Technology, can obtain with particular phenotype material as background covers wild rice full-length genome hybrid fragments introgressive line colony.Concrete steps are:
(1) Juvenile stage: donor parents is wild rice, receptor parent is black rice, and receptor parent General Requirements and wild rice genetic background have notable difference.
(2) parents: General Requirements receptor parent is maternal, and donor parents is paternal hybrid, the strain of hybridization individual plant general random 4.
(3) backcross: from F
1in generation, starts, and backcrosses, obtain BC1F1 about Stochastic choice 20 strain with receptor parent; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain 55 BC
2f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain the BC of 170
3f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and finally obtain 530 BC
4f
1strain; Next, then when running into a small amount of hybrid fragments lacked, be generally at BC
2f
1for reserve part strain, the later stage only screens with disappearance target label, is backcrossed by the individual plant containing disappearance target gene again.
(4) target label detects: first use 24 pairs of good primers of polymorphism, average every bar chromosome 2 pairs of primers, be distributed in chromosomal long-armed with on galianconism respectively, all individualities are analyzed, the individual plant of screening containing less than 1 or 1 hybrid fragments, then carries out full-length genome genotyping to the individual plant retained.
(5) selfing: after full-length genome genotyping is carried out to individual plant, choose the individual plant selfing having less than 2 hybrid fragments, and carry out bagging, then sowing.
(6) target label detects: selected single-strain planting is (because selected individual plant is BC
3f
2or BC
4f
2the seed in generation, will amplify colony as far as possible, could obtain the individual plant of target label), plant 200 strains containing 1 each strain of hybrid fragments, each strain containing 2 hybrid fragments plants 300 strains.
(7) hybrid fragments introgressive line individual plant screening: carry out detection with target label to plantation strain individual plant and analyze, (these individual plants are BC to select to contain target label heterozygosis individual plant
3f
3or BC
4f
3the seed in generation, does not need to plant again, because individual plant is heterozygosis individual plant); Also retain containing the target label individual plant that isozygotys simultaneously.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (2)
1. cover a construction method for wild rice full-length genome hybrid fragments introgressive line, it is characterized in that: it mainly comprises Juvenile stage, hybridizes, backcrosses, target label detection, selfing, target label detect; Concrete steps are as follows:
(1) Juvenile stage: donor parents is wild rice, receptor parent requires there is notable difference with wild rice genetic background;
(2) parents: receptor parent is maternal, and donor parents is male parent, the random 3-5 strain of hybridization individual plant;
(3) backcross: from F
1in generation, starts, and Stochastic choice 20-25 strain and receptor parent backcross, and obtain BC
1f
1; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain 55-65 BC
2f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and obtain the BC of 170-190
3f
1strain; The strain of each strain Stochastic choice 3 and receptor parent backcross, and finally obtain 530-550 BC
4f
1strain; Then when running into the hybrid fragments of disappearance, be at BC secondly,
2f
1for reserve part strain, the later stage only screens with disappearance target label, is backcrossed by the individual plant containing disappearance target gene again;
(4) target label detect: first with 23-25 to the good primer of polymorphism, average every bar chromosome 2 pairs of primers, be distributed in chromosomal long-armed with on galianconism respectively, all individualities are analyzed, the individual plant of screening containing less than 1 or 1 hybrid fragments, then carries out full-length genome genotyping to the individual plant retained;
(5) selfing: after full-length genome genotyping is carried out to individual plant, choose the individual plant selfing having less than 2 hybrid fragments, and carry out bagging, then sowing;
(6) target label detects: selected single-strain planting, containing 1 hybrid fragments each strain plantation 50-300 strain, and each strain plantation 100-400 strain containing 2 hybrid fragments;
(7) hybrid fragments introgressive line individual plant screening: with target label, detection is carried out to plantation strain individual plant and analyze, select containing target label heterozygosis individual plant; Also retain containing the target label individual plant that isozygotys simultaneously;
Selected individual plant in described step (6) is BC
3f
2or BC
4f
2the seed in generation; Heterozygosis individual plant in step (7) is BC
3f
3or BC
4f
3the seed in generation.
2. a kind of construction method covering wild rice full-length genome hybrid fragments introgressive line according to claim 1, is characterized in that: described receptor parent is long-grained nonglutinous rice or japonica rice.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1486593A (en) * | 2003-07-30 | 2004-04-07 | 上海市农业生物基因中心 | Construction process of rice genetic test material |
| CN101755674A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Breeding method of alien substitution line |
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| MX2014015429A (en) * | 2012-06-15 | 2015-07-14 | Agrigenetics Inc | Methods for selection of introgression marker panels. |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1486593A (en) * | 2003-07-30 | 2004-04-07 | 上海市农业生物基因中心 | Construction process of rice genetic test material |
| CN101755674A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Breeding method of alien substitution line |
Non-Patent Citations (2)
| Title |
|---|
| Construction of introgression lines carrying wild rice(Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits;Feng Tian, et al;《Theor Appl Genet》;20061231;第112卷;第570-580页，尤其是第573页第1栏第2段。 * |
| 覆盖野生稻全基因组染色体片段置换系构建进展;叶新福等;《中国农业科学》;20131231;第46卷(第24期);第5075-5080页，尤其是第5076页左栏第2段至右栏图，第5077页第5.1节至第5078页第5.4节。 * |
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