CN109486986A - A kind of japonica rice allusion quotation dye loses the efficient assisted selection method of restorer molecular labeling - Google Patents
A kind of japonica rice allusion quotation dye loses the efficient assisted selection method of restorer molecular labeling Download PDFInfo
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Abstract
The present invention discloses a kind of japonica rice allusion quotation dye and loses the efficient assisted selection method of restorer molecular labeling, including following operating procedure: (1) using first, ten chromosome restoring gene SSR of four parental rices of primer mark respectively, then it is expanded, electrophoresis obtains the restorer genotype of four parental rices；(2) by the intermolecular hybrid of four parental rices, F is then obtained2For group's restorer gene molecule marker assisted Selection as a result, in F2For the restorer swimming lane of molecular marker assisted selection in segregating population；(3) to F4In generation, carries out molecular marker assisted selection.The restorer strain number that molecular marker assisted selection is chosen in a short time is more, it is wide to restore type, saves human and material resources, financial resources.
Description
Technical field
The invention belongs to molecular labeling applied technical fields, and in particular to it is high that a kind of japonica rice allusion quotation dye loses restorer molecular labeling
Imitate assisted selection method.
Background technique
In recent years, molecular breeding is quickly grown, and remote edge kind or wild rice are successfully had niche in many researchs
Because importing excellent Rice Resources.Disease-resistant molecular breeding is the successful model of molecular marker assisted selection.It obtains from leaf blight
The new lines that horizontal and vertical resistance increases；Cultivate the bacterial blight-resisting restorer of a batch tool Xa-21 gene；In conjunction with anti-
The genetic map of rice blast gene Pi 25 newly increases molecular labeling RM3330 and A7 with the gene close linkage, and combines and divide
Sub- marker assisted selection as a result, and use the chain label in target gene two sides to carry out assisted Selection simultaneously, then can be obvious
Improve molecular marker assisted selection coincidence rate.
In grain quality rice breeding, molecular marker assisted selection also achieves many achievements, from control gelatinization temperature
The site insertion-deletion length polymorphism (InDel) is found in the gene coding region fgr of alk gene and control fragrance, and develops
InDel label carries out assisted selection.It is functional by comparing the fgr gene order and 9311 sequence designs of coding BAD2
STS marks Aro, and the molecular marker assisted selection for carrying out scent gene is marked using Aro.The molecule of rice restoring gene assists choosing
It selects at home still in the development phase, scientific research value with higher is innovative extremely strong scientific and technical method, is solution
The key technology of certainly current hybrid paddy rice development problem.
Existing technical disadvantages are to carry out selection to genotype indirectly by phenotype, theoretically, utilize molecular labeling
The difference of Direct Identification genotype therefore the disadvantage that phenotype infers genotype inaccuracy can be avoided from DNA level.Point
Sub- marker assisted selection overcomes the difficulty of trait expression type identification: the phenotype of some characters identifies extremely difficult, restorer
It can not identify and from table, difficulty is larger on measurement technology, program is cumbersome, the period is long, costly, low efficiency；Pass through
It is larger to be measured genotype and the gap of anticipation after identifying.
Summary of the invention
The object of the present invention is to provide a kind of japonica rice allusion quotation dyes to lose the efficient assisted selection method of restorer molecular labeling.
The present invention is achieved by the following technical solutions.
A kind of japonica rice allusion quotation dye loses the efficient assisted selection method of restorer molecular labeling, including following operating procedure:
(1) 422, R498, R527, C418 are taken turns with primer RM5629, four parental rice evenings of primer RM10353 label respectively
First, ten chromosome restoring gene SSR, then expanded, electrophoresis, obtain the restorer genotype of four parental rices,
The restorer genotype of R498 is Rf1Rf1Rf10Rf10, and the restorer genotype of R527 is Rf1Rf1Rf10Rf10, C418's
Restorer genotype is Rf10Rf10, and the restorer genotype of evening wheel 422 is Rf1Rf1Rf10Rf10；
(2) by the intermolecular hybrid evening wheel 422/R498 of four parental rices, evening wheel 422/R527, C418/R498, C418/
Then R527 obtains F2For group's restorer gene molecule marker assisted Selection as a result, in F2It is auxiliary for molecular labeling in segregating population
Help the restorer swimming lane of selection, genotype Rf1Rf1Rf10Rf10, therefore in F2For being selected in 22 strains in segregating population
The restorer of homozygous dominant restoring gene is had to 2 plants；
(3) to F4In generation, carries out molecular marker assisted selection, in F4For the restorer of molecular marker assisted selection in segregating population
Range expands, and has 18 plants with homozygous genotype Rf1Rf1Rf10Rf10, therefore in four generations compared with two generation segregating populations
Compared with efficiency of selection improves 9 times, wherein the sequence of RM5629 primer are as follows: AGCTCAACTCGAGAACTCCC, RM10353 primer
Sequence are as follows: TTGTTAGTTGGCGAAAGGAA.
Specifically, the specific extracting method of total DNA is as follows: (1) taking about 0.1g tender leaf to be put into 2ml centrifuge tube, be added
900 μ L of CTAB buffer is quickly smashed blade to pieces with no bacteria stick；It is taken out after (2) 65 DEG C of water-bath 30min, isometric chlorine is added
Imitative/isoamyl alcohol mixed liquor, is mixed by inversion 10 minutes；(3) 4 DEG C of centrifugations, 8000-12000rpm are centrifuged 10min；(4) supernatant is taken
The new centrifuge tube of another 1.5ml is moved to, the isopropanol being pre-chilled in equal volume is added, centrifuge tube is softly mixed into 5min；(5) 4 DEG C of centrifugations,
10000rpm is centrifuged 10min；(6) abandon supernatant, with after 70% ethanol washing 1 time again with 95% ethanol washing；(7) after air-drying,
Appropriate sterilized TE solution dilution is added, wherein CTAB buffer is 10ml 1mol/L Tris-HCl, 4ml0.5mol/L
EDTA, 10ml 5mol/L NaCl, add deionized water to be settled to 100ml, and TE solution is 1ml1mol/L Tris-HCl, 0.2ml
0.5mol/L EDTA, 98.8ml deionized water.
Specifically, PCR response procedures are as follows: initial denaturation 5min at 94 DEG C is denaturalized 30s at 94 DEG C, and anneal 30s, annealing temperature
It is 47 DEG C, 72 DEG C of extension 30s, totally 35 recycle, last 72 DEG C of extensions 7min；Amplified production 6% polyacrylamide gel electricity
Swimming takes 2 μ L of reaction product and 1 μ L sample-loading buffer to mix, and electrophoretic buffer is TBE solution, 120V constant pressure electrophoresis 1.5h, warp
0.05% AgNO3Solution dye 15 minutes, colour developing after carry out specific detection, wherein TBE solution is made of the following components:
54gTris alkali, 27.5g boric acid, 20ml 0.5mol/L EDTA, add water to be settled to 1000ml.
From the above technical scheme, it can be seen that the beneficial effects of the present invention are:
The present invention is auxiliary using molecular labeling mainly using North Japonica Rice and Indica Rice Restorer Lines filial generation separation material as group
The method for helping selection, screening selection have homozygous main effect restoring gene, show the good restorer of resume, select using gene
It selects and improves efficiency of selection, change and just can determine that the selection technique road with restoring gene by being measured crop field selection in the past
Line, the gene with excellent hereditary capacity are retained, and are also restorer introducing to solve the problems, such as that the friendship of Xian round-grained rice is not merged
Long-grained nonglutinous rice ingredient simultaneously makes Hybrid Japonica Rice have more good hybrid vigour.Rice genetic basic resource has been innovated simultaneously
Approach provides method and foundation for Germplasm enhancement, provides reliable base to expand Northern hybridization rice genetic background
Plinth guarantees.
Detailed description of the invention
Fig. 1 is 4 parent's restoring gene type molecular marker screenings of primer RM5629 amplification, and Fig. 2 is primer RM10353 expansion
The 4 parent's restoring gene type molecular marker screenings increased, M swimming lane are that D2000 DNA Ladder, A swimming lane is allusion quotation scum of a community type infertility
System, R swimming lane are restorer, and 1-4 swimming lane is four parents, are sequentially followed successively by R498, R527, C418, evening wheel 422.
Fig. 3 is 22 strains in two generation segregating populations of primer RM5629 amplification, and Fig. 4 is primer RM10353 amplification
22 strains in two generation segregating populations, M are D2000 DNA Ladder；1 swimming lane is novel sterile line, and 2 swimming lanes are restorer,
3-24 swimming lane is 22 strains in segregating population.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products of commercially available acquisition can be passed through.
Embodiment 1
A kind of japonica rice allusion quotation dye loses the efficient assisted selection method of restorer molecular labeling, including following operating procedure:
(1) 422, R498, R527, C418 are taken turns with primer RM5629, four parental rice evenings of primer RM10353 label respectively
First, ten chromosome restoring gene SSR, then expanded, electrophoresis, obtain the restorer genotype of four parental rices,
The restorer genotype of R498 is Rf1Rf1Rf10Rf10, and the restorer genotype of R527 is Rf1Rf1Rf10Rf10, C418's
Restorer genotype is Rf10Rf10, and the restorer genotype of evening wheel 422 is Rf1Rf1Rf10Rf10；
(2) by the intermolecular hybrid evening wheel 422/R498 of four parental rices, evening wheel 422/R527, C418/R498, C418/
Then R527 obtains F2For group's restorer gene molecule marker assisted Selection as a result, in F2It is auxiliary for molecular labeling in segregating population
Help the restorer swimming lane of selection, genotype Rf1Rf1Rf10Rf10, therefore in F2For being selected in 22 strains in segregating population
The restorer of homozygous dominant restoring gene is had to 2 plants；
(3) to F4In generation, carries out molecular marker assisted selection, in F4For the restorer of molecular marker assisted selection in segregating population
Range expands, and has 18 plants with homozygous genotype Rf1Rf1Rf10Rf10, therefore in four generations compared with two generation segregating populations
Compared with efficiency of selection improves 9 times, wherein the sequence of RM5629 primer are as follows: AGCTCAACTCGAGAACTCCC, RM10353 primer
Sequence are as follows: TTGTTAGTTGGCGAAAGGAA.
Specifically, the specific extracting method of total DNA is as follows: (1) taking about 0.1g tender leaf to be put into 2ml centrifuge tube, be added
900 μ L of CTAB buffer is quickly smashed blade to pieces with no bacteria stick；It is taken out after (2) 65 DEG C of water-bath 30min, isometric chlorine is added
Imitative/isoamyl alcohol mixed liquor, is mixed by inversion 10 minutes；(3) 4 DEG C of centrifugations, 8000-12000rpm are centrifuged 10min；(4) supernatant is taken
The new centrifuge tube of another 1.5ml is moved to, the isopropanol being pre-chilled in equal volume is added, centrifuge tube is softly mixed into 5min；(5) 4 DEG C of centrifugations,
10000rpm is centrifuged 10min；(6) abandon supernatant, with after 70% ethanol washing 1 time again with 95% ethanol washing；(7) after air-drying,
Appropriate sterilized TE solution dilution is added, wherein CTAB buffer is 10ml 1mol/L Tris-HCl, 4ml0.5mol/L
EDTA, 10ml 5mol/L NaCl, add deionized water to be settled to 100ml, and TE solution is 1ml1mol/L Tris-HCl, 0.2ml
0.5mol/L EDTA, 98.8ml deionized water.
Specifically, PCR response procedures are as follows: initial denaturation 5min at 94 DEG C is denaturalized 30s at 94 DEG C, and anneal 30s, annealing temperature
It is 47 DEG C, 72 DEG C of extension 30s, totally 35 recycle, last 72 DEG C of extensions 7min；Amplified production 6% polyacrylamide gel electricity
Swimming takes 2 μ L of reaction product and 1 μ L sample-loading buffer to mix, and electrophoretic buffer is TBE solution, 120V constant pressure electrophoresis 1.5h, warp
0.05% AgNO3Solution dye 15 minutes, colour developing after carry out specific detection, wherein TBE solution is made of the following components:
54g Tris alkali, 27.5g boric acid, 20ml 0.5mol/L EDTA, add water to be settled to 1000ml.
There is no identical banding pattern with A swimming lane in attached drawing 1, banding pattern identical as R swimming lane there are 1,2,3,4 swimming lanes, is restorer, contains
There is Rf10 homozygous gene.Banding pattern identical as A swimming lane has 3 swimming lanes in B figure, is free of Rf1 homozygous gene；Banding pattern identical as R swimming lane
Have 1,2,4 swimming lanes, contain Rf1 homozygous gene.In conclusion the restorer genotype of R498 is Rf1Rf1Rf10Rf10,
The restorer genotype of R527 is Rf1Rf1Rf10Rf10, and the restorer genotype of C418 is Rf10Rf10, the recovery of evening wheel 422
Be genotype be Rf1Rf1Rf10Rf10.Evening wheel 422, R498, R527 have restoring gene sun on first, ten chromosomes in parent
Property homozygote (RfRf), C418 only have restoring gene positive homozygote (RfRf) on ten chromosomes, do not have the first chromosome
Upper restoring gene positive homozygote (RfRf), and do not find restoring gene heterozygote (Rfrf), recessive homozygosis in four parents
Body (rfrf).Illustrate that this four parents are the stable materials of genotype, evening wheel 422, R498, R527, which have, to be restored to compose wide spy
Point extensive allusion quotation can lose and contaminate the sterile line of scum of a community's type simultaneously, and C418 can only restore the sterile line of dye scum of a community's type.
Banding pattern identical as 1 swimming lane has 12,13 swimming lanes in attached drawing 3, is sterile plant, genotype rf10rf10；With 2 swimming lanes
Identical banding pattern has 4,6,8,9,11,14,17,24 swimming lanes, is fertile strain, genotype Rf10Rf10, remaining swimming lane gene
Type is Rf10rf10.Banding pattern identical as 1 swimming lane has 5,6,10,13,17,18,20,22 swimming lanes in B figure, is sterile plant, gene
Type is rf1rf1；Banding pattern identical as 2 swimming lanes has 3,9,11 swimming lanes, is fertile plant, genotype Rf1Rf1；Remaining swimming lane gene
Type is Rf1rf1.In conclusion in two generation segregating populations molecular marker assisted selection restorer swimming lane 9,11, genotype
Rf1Rf1Rf10Rf10, therefore 2 plants are chosen in 22 strains in two generation segregating populations with homozygous dominant restoring gene
Restorer.
1 four combination F2 of table are for molecular labeling restoring gene frequency
To the F of each combination plant2In generation, carries out molecular marker assisted selection, the frequency that discovery restoring gene list heterozygous occurs
Rate is higher, and the frequency highest that double heterozygous occur, the frequency of homozygous appearance is minimum, in conjunction with pollen microscopic examination as a result, choosing normal
Pollen grain proportion is more than 70% plant progress test cross, chooses 762 plants altogether, is analyzed by being measured setting percentage, is sent out
The setting percentage of the cross combination setting percentage highest of the genotype of existing phenotype homozygosis, single heterozygote missing Rf10 is higher, single miscellaneous
The setting percentage of zoarium missing Rf1 is lower, and the setting percentage of double heterozygote missing Rf1Rf10 is minimum.Illustrate to lack in double heterozygote
Dominant restoring gene setting percentage is influenced it is maximum, but heterozygote lack dominant restoring gene one of both on setting percentage influence compared with
Greatly, but the influence of missing Rf1 becomes apparent, and phenotype homozygosis restoring gene is the emphasis of restorer molecular marker assisted selection,
Reliable technical guarantee is provided to cultivate restorer.
2 four combination F2 of table are for pollen staining and are measured setting percentage
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the art
Those of ordinary skill, within the essential scope of the present invention, variation, change, addition or the replacement made all should belong to the present invention
Protection scope.
Claims (3)
1. a kind of japonica rice allusion quotation dye loses the efficient assisted selection method of restorer molecular labeling, which is characterized in that walked including following operation
It is rapid:
(1) respectively with primer RM5629, primer RM10353 label four parental rice evenings wheel 422, R498, R527, C418 the
One, ten chromosome restoring gene SSR, is then expanded, electrophoresis, and the restorer genotype of four parental rices, R498 are obtained
Restorer genotype be Rf1Rf1Rf10Rf10, the restorer genotype of R527 is Rf1Rf1Rf10Rf10, the recovery of C418
It is genotype for Rf10Rf10, the restorer genotype of evening wheel 422 is Rf1Rf1Rf10Rf10；
(2) by the intermolecular hybrid evening wheel 422/R498 of four parental rices, evening wheel 422/R527, C418/R498, C418/R527, so
After obtain F2For group's restorer gene molecule marker assisted Selection as a result, in F2For molecular marker assisted selection in segregating population
Restorer swimming lane, genotype Rf1Rf1Rf10Rf10, therefore in F2For choosing 2 plants of bands in 22 strains in segregating population
There is the restorer of homozygous dominant restoring gene；
(3) to F4In generation, carries out molecular marker assisted selection, in F4For the restorer range of molecular marker assisted selection in segregating population
It expands, has 18 plants with homozygous genotype Rf1Rf1Rf10Rf10, therefore in four generations compared with two generation segregating populations, choosing
It selects efficiency and improves 9 times；
The wherein sequence of RM5629 primer are as follows: the sequence of AGCTCAACTCGAGAACTCCC, RM10353 primer are as follows:
TTGTTAGTTGGCGAAAGGAA。
2. a kind of japonica rice allusion quotation dye according to claim 1 loses the efficient assisted selection method of restorer molecular labeling, feature
It is, the specific extracting method of total DNA is as follows: (1) about 0.1g tender leaf is taken to be put into 2ml centrifuge tube, CTAB buffer 900 is added
μ L is quickly smashed blade to pieces with no bacteria stick；It is taken out after (2) 65 DEG C of water-bath 30min, isometric chloroform/isoamyl alcohol mixing is added
Liquid is mixed by inversion 10 minutes；(3) 4 DEG C of centrifugations, 8000-12000rpm are centrifuged 10min；(4) supernatant is taken to move to another 1.5ml
New centrifuge tube is added the isopropanol being pre-chilled in equal volume, centrifuge tube is softly mixed 5min；(5) 4 DEG C of centrifugations, 10000rpm centrifugation
10min；(6) abandon supernatant, with after 70% ethanol washing 1 time again with 95% ethanol washing；(7) after air-drying, addition has been gone out in right amount
The TE solution of bacterium dilutes, and wherein CTAB buffer is 10ml 1mol/L Tris-HCl, 4ml 0.5mol/L EDTA, 10ml
5mol/L NaCl, adds deionized water to be settled to 100ml, and TE solution is 1ml 1mol/L Tris-HCl, 0.2ml 0.5mol/L
EDTA, 98.8ml deionized water.
3. a kind of japonica rice allusion quotation dye according to claim 1 loses the efficient assisted selection method of restorer molecular labeling, feature
It is, PCR response procedures are as follows: initial denaturation 5min at 94 DEG C is denaturalized 30s at 94 DEG C, and anneal 30s, and annealing temperature is 47 DEG C, 72 DEG C
Extend 30s, totally 35 circulations, last 72 DEG C of extensions 7min；6% polyacrylamide gel electrophoresis of amplified production, negating should produce
2 μ L of object and 1 μ L electrophoretic buffer mix, and electrophoretic buffer is TBE solution, 120V constant pressure electrophoresis 1.5h, through 0.05% AgNO3
Solution dye 15 minutes, colour developing after carry out specific detection, wherein TBE solution is made of the following components: 54gTris alkali, 27.5g
Boric acid, 20ml 0.5mol/L EDTA, add water to be settled to 1000ml.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1831138A (en) * | 2005-11-21 | 2006-09-13 | 安徽省农业科学院水稻研究所 | Molecular marking supplementary breeding method for multi-purpose restoring series of three-series hybrid rice |
| CN101485283A (en) * | 2008-12-29 | 2009-07-22 | 重庆市农业科学院生物技术研究中心 | Method for improving selection efficiency in hybrid rice breeding |
| WO2011090690A1 (en) * | 2009-12-28 | 2011-07-28 | Pioneer Hi-Bred International, Inc. | Sorghum fertility restorer genotypes and methods of marker-assisted selection |
| CN106566888A (en) * | 2016-11-08 | 2017-04-19 | 淮阴师范学院 | Method for selecting and breeding variety with diversified resistance by molecular marker-assisted selection |
| CN107385024A (en) * | 2017-07-04 | 2017-11-24 | 华智水稻生物技术有限公司 | Rice fertility restorer genes assistant breeding molecular labeling and its application |
-
2018
- 2018-09-14 CN CN201811072234.1A patent/CN109486986B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1831138A (en) * | 2005-11-21 | 2006-09-13 | 安徽省农业科学院水稻研究所 | Molecular marking supplementary breeding method for multi-purpose restoring series of three-series hybrid rice |
| CN101485283A (en) * | 2008-12-29 | 2009-07-22 | 重庆市农业科学院生物技术研究中心 | Method for improving selection efficiency in hybrid rice breeding |
| WO2011090690A1 (en) * | 2009-12-28 | 2011-07-28 | Pioneer Hi-Bred International, Inc. | Sorghum fertility restorer genotypes and methods of marker-assisted selection |
| CN106566888A (en) * | 2016-11-08 | 2017-04-19 | 淮阴师范学院 | Method for selecting and breeding variety with diversified resistance by molecular marker-assisted selection |
| CN107385024A (en) * | 2017-07-04 | 2017-11-24 | 华智水稻生物技术有限公司 | Rice fertility restorer genes assistant breeding molecular labeling and its application |
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