CN105648091A - Molecular marker-assisted selection method for transferring excellent rape thermo-sensitive male sterile line - Google Patents
Molecular marker-assisted selection method for transferring excellent rape thermo-sensitive male sterile line Download PDFInfo
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- CN105648091A CN105648091A CN201610144773.6A CN201610144773A CN105648091A CN 105648091 A CN105648091 A CN 105648091A CN 201610144773 A CN201610144773 A CN 201610144773A CN 105648091 A CN105648091 A CN 105648091A
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Abstract
The invention provides a molecular marker-assisted selection method for transferring an excellent rape thermo-sensitive male sterile line. The molecular marker-assisted selection method is characterized in that a CTAB method is used to extract the genome DNA of the filial generation single plant of an alternative excellent rape thermo-sensitive male sterile line, and agarose gel electrophoresis is used to detect the quality of the DNA sample; an interlocking SSR marker is used to perform PCR amplification on the genome DNA of the filial generation single plant, and the forward primer sequence and reverse primer sequence of the interlocking SSR marker are respectively 5'-CTCTCCTGATCCTCTCCTTC-3' and 5'-CTTGTAGAGAACCCGAACTG-3'; 6% polyacrylamide gel is used to perform electrophoresis detection on the PCR amplification product, and the plant with the target band is selected according to the electrophoresis detection result as the breeding material of the excellent rape thermo-sensitive male sterile line.
Description
Technical field
The present invention relates to field of molecular breeding, be specifically related to a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation.
Background technology
Brassica campestris L temp-sensing sterile line has that group joins freedom, Restorer varieties is wide, breeding cycle is short, easily selects the advantages such as influential point set, accordingly, as the new way that rape heterosis utilizes, has broad application prospects. Brassica campestris L temp-sensing sterile line K121S belongs to mustard type rape, and the rape variety cultivated at present is cabbage type rape, belong to different species, in order to utilize this Brassica campestris L temp-sensing sterile line transformation to go out excellent cabbage type rape temp-sensing sterile line, require over intervarietal hybridization by the sterile gene of K121S and fertility-altercation gene transformation to cabbage type rape, then pass through backcross and artificial selection select excellent sterile system, but the artificial selection in general land for growing field crops is subject to seasonal restrictions, and time-consuming take a lot of work, efficiency of selection is relatively low. And molecular marker assisted selection has the selection advantages such as purposiveness is strong, efficiency of selection is high, the selection time is free, it is widely used in the artificial selection of breeding material.
Summary of the invention
In order to solve problems of the prior art, the invention provides a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation.
A kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation of the present invention, including comprising the following steps:
Step is 1.: adopts CTAB method to extract the genomic DNA of the filial generation individual plant for excellent Brassica campestris L temp-sensing sterile line selection-breeding, and detects the quality of DNA sample;
Step is 2.: with step described in chain SSR marker pcr amplification 1. in the individual plant genomic DNA that extracts, and with the polyacrylamide gel electrophoresis detection amplified production of 4%��8%. Forward and reverse primer sequence of chain SSR marker is 5 '-CTCTCCTGATCCTCTCCTTC-3 ' and 5 '-CTTGTAGAGAACCCGAACTG-3 ' respectively.
Step is 3.: according to step 2. in electrophoresis detection result, will have size and be about the plant breeding material as the excellent further selection-breeding of Brassica campestris L temp-sensing sterile line of 550bp object tape.
A kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation of the present invention, step 2. in pcr amplification reaction system be: 10 �� PCR buffer 1.0��3.0 �� l, concentration is the MgCl of 25mmol/L2Solution 2.0��3.0 �� l, concentration is deoxynucleoside acid solution 0.3��0.7 �� l of 2.5mmol/L, and concentration is Taq enzyme 0.7��0.9 �� l of 5U/ �� l, and concentration is forward and reverse primer each 0.3��0.7 �� l, the DNA of 2��6 �� l of 10mmol/L, adds ddH2O to 10.0��30.0 �� l;The linkage distance of step 2. middle linked marker and target gene is 0.8��1cm.
A kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation of the present invention, step 2. in pcr amplification program be: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, 35 circulations; 72 DEG C of ends extend 10min, 4 DEG C of preservations.
In order to make pcr amplification result more preferably, the described step 1. middle genomic DNA extracted needs to detect its DNA content and integrity, detection method is at 0.8% agarose gel by genome DNA sample, 0.5 �� tbe buffer liquid, electrophoresis under 3-5V/cm, described agarose gel adds nucleic acid dye Goldview, detects content and the integrity of described genome DNA sample after the photograph of electrophoresis result gel imaging system, and utilize the absorbance of nucleic acid-protein detector detection genome DNA sample.
The present invention has the advantage that relative to prior art
1, the present invention carries out molecular marker assisted selection when the excellent Brassica campestris L temp-sensing sterile line of transformation by this SSR marker, improves selection purposiveness, is about the Single-plant selection of 550bp object tape out by having size, as the breeding material of excellent temp-sensing sterile line transformation.
2, the present invention carries out molecular marker assisted selection when the excellent Brassica campestris L temp-sensing sterile line of transformation by this SSR marker, improves efficiency of selection, decreases the input of manpower and materials, has saved the time selected.
3, the present invention carries out molecular marker assisted selection when the excellent Brassica campestris L temp-sensing sterile line of transformation by this SSR marker, improves selection degree of freedom, overcomes the land for growing field crops artificial selection restriction by planting season, can be carried out any time selecting.
4, in the present invention, linked marker is very near with the linkage distance of target gene, only 0.8��1cm, and chain is very close, as long as the plant amplifying object tape just has this character, and can as by roguing.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result figure of chain primer PCR amplification individual plant DNA.
Detailed description of the invention
Below in conjunction with accompanying drawing, a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation of the present invention is described in further detail.
Fig. 1 show the electrophoresis result figure of chain primer PCR amplification individual plant DNA, and the individual plant with object tape (about 550bp) is alternative individual plant.
A kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation of the present invention comprises the following steps:
Step is 1.: adopt CTAB method to extract the genomic DNA of the filial generation individual plant for excellent Brassica campestris L temp-sensing sterile line selection-breeding, and detect the quality of DNA sample, detection method is at 0.8% agarose gel by genome DNA sample, 0.5 �� tbe buffer liquid, electrophoresis under 3-5V/cm, agarose gel adds Goldview, detect size and the integrity of genome DNA sample after the photograph of electrophoresis result gel imaging system, utilize the absorbance of nucleic acid-protein detector detection genome DNA sample simultaneously;
Step is 2.: with chain molecular marker pcr amplification step 1. in the genomic DNA that extracts, upstream and downstream the sequence respectively 5 '-CTCTCCTGATCCTCTCCTTC-3 ' and 5 '-CTTGTAGAGAACCCGAACTG-3 ' of primer during pcr amplification, detect amplified production by the polyacrylamide gel electrophoresis of 6% after pcr amplification;
Step is 3.: according to step 2. in electrophoresis detection result, as it is shown in figure 1, size will be had be about the plant breeding material as the excellent further selection-breeding of Brassica campestris L temp-sensing sterile line of 550bp object tape.
Wherein, pcr amplification reaction system is: 10 �� PCR buffer 2.0 �� l, and concentration is the MgCl of 25mmol/L2Solution 2.5 �� l, concentration is the deoxynucleoside acid solution 0.5 �� l of 2.5mmol/L, and concentration is the Taq enzyme 0.8 �� l of 5U/ �� l, and concentration is forward and reverse primer each 0.5 �� l, the DNA of 4 �� l of 10mmol/L, adds ddH2O to 20.0 �� l.
Pcr amplification program is: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, 35 circulations; 72 DEG C of ends extend 10min, 4 DEG C of preservations.
A kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation of the present invention, experimental implementation process specifically includes that the breeding material obtaining alternative, extract the genomic DNA of individual plant, utilize the DNA of chain molecular marker (SSR primer) each individual plant of pcr amplification, detected through gel electrophoresis amplified production, using the individual plant with object tape as selecting result.
1, extracting genome DNA (CTAB method)
(1) weigh the blade that 1.0g children is tender, blade is put in the mortar of Liquid nitrogen precooler, and is milled to rapidly powder;
(2) ground blade powder is proceeded in 1.5ml centrifuge tube, and add the CTAB600ul of 65 DEG C of preheatings;
(3) water-bath being adjusted to 65 DEG C of constant temperature, put into the centrifuge tube equipped with CTAB and sample, rock centrifuge tube every 10-15min and make solution mix, water-bath is about 1h;
(4) from water-bath, take out centrifuge tube, after its cooling, add the chloroform of equal-volume (about 600ul): the mixed liquor of isoamyl alcohol=24:1 also mixes gently, and 12000rpm is centrifuged 15min;
(5) take centrifugal after supernatant, and load in a new centrifuge tube;
(6) step (4) is repeated;
(7) taking supernatant, proceed in a new centrifuge tube, add the isopropanol of isopyknic-20 DEG C of pre-coolings, mixing is placed at-20 DEG C of temperature 3-4 hour, and the centrifugal 10min of 12000rpm abandons supernatant;
(8) adding the ethanol of 500ul75%, washing precipitation in centrifuge tube, at 4 DEG C, the centrifugal 5min of 8000rpm, abandons supernatant, washs 2-3 time, is placed in absorbent paper and is inverted dries;
(9) 50ulTE buffer solution DNA is added, standby in-20 DEG C.
2, the detection of genomic DNA
DNA sample, at 0.8% agarose gel, 0.5 �� tbe buffer liquid, electrophoresis under 3-5V/cm, adds Goldview in gel, electrophoresis result gel imaging system is taken a picture, and detects DNA sample size and integrity, utilizes nucleic acid-protein detector detection absorbance.
3, pcr amplification reaction system
10 �� PCRBuffer2.0 �� l, 25mmol/LMgCl2The Taq enzyme 0.8 �� l of 2.5 �� l, 2.5mmol/LdNTPs0.5 �� l, 5U/ �� l, concentration is forward and reverse primer each 0.5 �� l, the 4 �� lDNA of 10mmol/L, adds ddH2O to 20.00 �� l.
Amplification program is: 94 DEG C of denaturation 5min; 94 DEG C of 1min, 54 DEG C of 1min, 72 DEG C of 1.5min, 35 circulations; 72 DEG C of ends extend 10min, 4 DEG C of preservations.
4, pcr amplification product polyacrylate hydrogel electrophoresis
By 6% Polyacrylamide Gel Electrophoresis PCR primer, 6% polyacrylamide gel preparation process is as follows:
(1) clean glass plate and comb with tap water, then use deionized water fully to rinse, finally with 75% ethanol hydro-peening, dry with chipless napkin;
(2) glued membrane edge preventing from cementing leakage is sealed with the agarose of melted 2%;
(3) 6% polyacrylamide gel are now with the current;
(4) to encapsulating in rubber moulding, when glue to short glass plate edge, stop encapsulating, process is avoided produce bubble;
(5) insert comb, make glue stand fully polymerization 60��90min;
(6) by 0.5 �� TBE electrophoretic buffer cleaning down loading wells;
(7) rubber moulding is put into electrophoresis tank, in upper and lower electrophoresis tank, pour the 0.5 �� TBE electrophoretic buffer newly joined in right amount respectively into;
(8) comb is vertically extracted, by microsyringe correction loading wells.
Prerunning 30min, setting power is 50W; After electrophoresis terminates, in loading wells, add the amplified production 6.0 �� L of mixed sample-loading buffer, when to be instructed dose of electrophoresis is to gel leading edge 15cm place, stop electrophoresis.
5, silver dye
(1) fixing: the long glass plate being stained with gel is immersed 1%HNO3In solution, being shaken gently for, fixing 5min, deionization is washed 2 times, takes out glass plate;
(2) gel is immersed dyeing liquor (0.1%AgNO3Solution, by front addition formalin to 0.75%), it is shaken gently for, lucifuge dyeing 12min;
(3) deionization is washed 2 times, and (first time, direct deionized water washed 30s to turn-over; Second time adds 120 �� l10%Na in deionized water2SO3Wash 30s);
(4) gel is immersed the developer solution (3.0%Na of 4 DEG C of pre-coolings2CO3Solution, by front addition formalin to 0.02%), jog is until seeing band colour developing until aobvious band appropriateness, and deionization is washed 1 time;
(5) fixative adding recovery terminates reaction 1min, again washes 1 time with deionized water, scans blob of viscose record electrophoresis result.
6, the presence or absence according to object tape, it is determined that selected plant.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all any amendment, equivalent replacement and improvement etc. made within the spirit and principles in the present invention, should be included within protection scope of the present invention.
Claims (6)
1. the molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation, it is characterised in that comprise the following steps:
Step is 1.: adopts CTAB method to extract the genomic DNA of the filial generation individual plant for excellent Brassica campestris L temp-sensing sterile line selection-breeding, and detects the quality of DNA sample;
Step is 2.: with step described in chain SSR marker pcr amplification 1. in the genomic DNA that extracts, and with the polyacrylamide gel electrophoresis detection amplified production of 4%��8%;
Step is 3.: according to described step 2. in electrophoresis detection result, using breeding material as the excellent further selection-breeding of Brassica campestris L temp-sensing sterile line of the plant with object tape.
2. a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation as claimed in claim 1, it is characterized in that, during described step 2. middle pcr amplification, forward and reverse primer sequence of chain SSR marker is 5 '-CTCTCCTGATCCTCTCCTTC-3 ' and 5 '-CTTGTAGAGAACCCGAACTG-3 ' respectively.
3. a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation as claimed in claim 2, it is characterised in that described step 2. in the linkage distance of linked marker and target gene be 0.8��1cm.
4. a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation as claimed in claim 3, it is characterised in that described step 2. in pcr amplification reaction system be: 10 �� PCR buffer 1.0��3.0 �� l, concentration is the MgCl of 25mmol/L2Solution 2.0��3.0 �� l, concentration is deoxynucleoside acid solution 0.3��0.7 �� l of 2.5mmol/L, and concentration is Taq enzyme 0.7��0.9 �� l of 5U/ �� l, and concentration is forward and reverse primer each 0.3��0.7 �� l, the DNA of 2��6 �� l of 10mmol/L, adds ddH2O to 10.0��30.0 �� l.
5. a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation as claimed in claim 4, it is characterised in that described step 2. in pcr amplification program be: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, 35 circulations; 72 DEG C of ends extend 10min, 4 DEG C of preservations.
6. a kind of molecular marker-assisted selection method for the excellent Brassica campestris L temp-sensing sterile line of transformation as claimed in claim 5, it is characterized in that, described step 3. in object tape size is about the plant breeding material as the excellent further selection-breeding of Brassica campestris L temp-sensing sterile line of 550bp.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102321767A (en) * | 2011-10-18 | 2012-01-18 | 湖南省作物研究所 | Simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102321767A (en) * | 2011-10-18 | 2012-01-18 | 湖南省作物研究所 | Simple sequence repeat-polymerase chain reaction (SSR-PCR)-based hybrid rape seed purity detection method |
Non-Patent Citations (2)
| Title |
|---|
| LOWE,T.M等: "XM_013794070", 《GENBANK》 * |
| 周筱妍等: "油菜温敏雄性不育系K121S育性转换基因的分子定位", 《分子植物育种》 * |
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