CN101487049A - Method for eliminating pepper epidemic disease breeding material by using molecular marker - Google Patents
Method for eliminating pepper epidemic disease breeding material by using molecular marker Download PDFInfo
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- 238000009395 breeding Methods 0.000 title claims abstract description 83
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- 201000010099 disease Diseases 0.000 title claims abstract description 64
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 64
- 235000002566 Capsicum Nutrition 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000006002 Pepper Substances 0.000 title claims abstract description 41
- 235000016761 Piper aduncum Nutrition 0.000 title claims abstract description 41
- 235000017804 Piper guineense Nutrition 0.000 title claims abstract description 41
- 235000008184 Piper nigrum Nutrition 0.000 title claims abstract description 41
- 244000203593 Piper nigrum Species 0.000 title 1
- 239000003147 molecular marker Substances 0.000 title 1
- 241000722363 Piper Species 0.000 claims abstract description 40
- 238000012408 PCR amplification Methods 0.000 claims abstract description 26
- 238000001962 electrophoresis Methods 0.000 claims abstract description 19
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960005542 ethidium bromide Drugs 0.000 claims abstract description 6
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- 238000000926 separation method Methods 0.000 claims abstract description 6
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- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
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- 229920002684 Sepharose Polymers 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
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- 238000009331 sowing Methods 0.000 abstract 1
- 241000208293 Capsicum Species 0.000 description 20
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- 230000009182 swimming Effects 0.000 description 8
- 241000233614 Phytophthora Species 0.000 description 6
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Abstract
The invention discloses a method for eliminating pepper epidemic disease breeding materials by using molecular markers, which comprises the steps of sowing pepper breeding materials with unknown epidemic disease resistance in a seedling tray for conventional management, extracting genome DNA of tender leaves of different breeding materials by using an improved SDS method when seedlings grow to 3-4 true leaves, and performing PCR amplification by using forward and reverse primers of the molecular markers. And carrying out electrophoretic separation on the PCR amplification product on 1.2% agarose gel, staining by ethidium bromide, observing by using a gel imaging system, and recording an electrophoresis result. If the electrophoresis result shows that a characteristic strip with the length of 990bp can be amplified, the material is a pepper breeding material with epidemic diseases and is eliminated; if the electrophoresis result shows that a characteristic strip with the length of 990bp can not be amplified, the material is an anti-epidemic disease pepper breeding material. The method can eliminate pepper epidemic disease infected breeding material in early growth stage, and has the advantages of short elimination period, low cost, reliable result, etc., and does not affect the observation of other characters.
Description
Technical field
The invention belongs to field of plant breeding, relate to a kind of superseded method of capsicum sense epidemic disease resistant breeding material, particularly a kind of method of utilizing the molecule marker culling pepper epidemic disease resistant breeding material.Utilizing this method capsicum can be felt epidemic disease resistant breeding material is eliminated at the commitment of its growth, have short, advantages such as cost is low, reliable results of cycle, and do not influence observation, can save great amount of manpower and material resources and land resources, and improve breeding efficiency greatly other proterties.
Technical background
Capsicum epidemic disease is a kind of crushing soil-borne disease that is caused by phytophthora (Phytophthora capsici L.), all over the world generation is arranged all, and then there is the trend that increases the weight of year by year in China.When this disease was very popular, the field capsicum was wilted in flakes until whole death, causes the output of capsicum sharply to reduce, even total crop failure.
Seed selection capsicum blight-resistant kind is one of control this disease most economical valid approach, but both there had been vertical resistance in capsicum to the resistance of eqpidemic disease, also had horizontal resistance, in some breeding material, and vertical resistance and horizontal resistance coexistence.In addition, different pepper breeding materials present the characteristics of " heredity variation " to the resistance of eqpidemic disease, show as monogenic inheritance, oligogenic inheritance and polygenic inheritance coexistence.Screen like this, directly that the breeding for disease resistance material carries out that the capsicum blight-resistant breeding of new variety will exist that qualification result is inaccurate, breeding cycle is long and many limitations such as new variety resistance instability.
It is the another kind of feasible way of capsicum blight-resistant breeding that susceptible breeding material is eliminated, not only can avoid the problem of capsicum blight-resistant evaluation difficulty, and can keep the genetic diversity of breeding for disease resistance material to greatest extent, satisfy the realization of other different breeding objectives to greatest extent.
The main method of the culling pepper epidemic disease resistant breeding material that uses is artificial inoculation in seedling stage at present, this method complex operation, easily affected by environment, and qualification result is inaccurate, unstable.Utilize that molecule marker carries out capsicum sense epidemic disease resistant breeding material superseded have simple to operate, reliable results, not affected by environment, advantage such as appraisal cost is low.The contriver utilizes the cDNA-AFLP technology to obtain behind the inoculation phytophthora gene of special abduction delivering in capsicum sense epidemic disease resistant breeding material, can obtain to feel the molecule marker of eqpidemic disease pepper breeding material according to the nucleotide sequence design special primer of this gene, to be used to feeling eliminating fast of eqpidemic disease pepper breeding material, accelerate the seed selection process of the good new variety of capsicum blight-resistant.
Summary of the invention
Problem at existing in the anti-eqpidemic disease evaluation of above-mentioned pepper breeding material the objective of the invention is to, and proposes a kind of method of utilizing the molecule marker culling pepper epidemic disease resistant breeding material.This method utilizes molecular marking technique to eliminate the pepper breeding material of sense eqpidemic disease at the growth commitment, have short, advantages such as cost is low, reliable results of cycle, and do not influence observation, can save great amount of manpower and material resources and land resources, and improve breeding efficiency greatly other proterties.
To achieve these goals, the present invention adopts following technical solution:
A kind of method of utilizing the molecule marker culling pepper epidemic disease resistant breeding material is characterized in that detailed process comprises the steps:
(1) will resist the pepper breeding material of eqpidemic disease the unknown to be seeded in seedling pan and carry out Routine Management, when treating that seedling grows to 3～4 true leaves, extract the genomic dna of different breeding material young leaflet tablets respectively with improvement SDS method;
(2) genomic dna with above-mentioned young leaflet tablet is a template, utilizes forward and reverse primer of molecule marker to carry out pcr amplification reaction;
The nucleotides sequence of the forward primer of the used molecule marker of above-mentioned pcr amplification reaction is classified 5 '-ctgtgatatggaaggggaacc-3 ' as, and the nucleotides sequence of reverse primer is classified 5 '-aactcgctgggaaaagaatg-3 ' as.
The cumulative volume of above-mentioned pcr amplification reaction is 25 μ L, wherein contains 1 * amplification Buffer, 10ng～25ng template DNA, the MgCl of 2.0mmol/L
2, the forward primer of 0.4 μ mol/L, the reverse primer of 0.4 μ mol/L, the dNTPs of 0.2mmol/L, 0.5U Taq archaeal dna polymerase.
The program of above-mentioned pcr amplification reaction is: 95 ℃ of pre-sex change 5min, enter the PCR circulation again, and cycling program is 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 34 circulations altogether, extend 10min, 4 ℃ of preservations down at 72 ℃ at last.
(3) pcr amplification product carries out electrophoretic separation on 1.2% sepharose, and ethidium bromide staining utilizes gel imaging system to observe, write down electrophoresis result;
(4) electrophoresis result is carried out following judgement:
If the feature band that it is 990bp that the electrophoresis result demonstration can amplify a length illustrates this breeding material for sense eqpidemic disease pepper breeding material, and it is superseded;
If the feature band that it is 990bp that the electrophoresis result demonstration can not amplify a length illustrates that this breeding material is anti-eqpidemic disease pepper breeding material, can be used for further capsicum blight-resistant breeding of new variety.
Seedling stage, compared by inoculation method with manually for method of the present invention, has following technique effect:
(1) utilizes method culling pepper epidemic disease resistant breeding material of the present invention, can carry out, and do not influence observation other proterties at the commitment of breeding material growth.
(2) utilize method culling pepper epidemic disease resistant breeding material of the present invention, the reliability height is not influenced by external environmental condition.
(3) utilize method culling pepper epidemic disease resistant breeding material of the present invention, the required cycle is short, can obtain The selection result in 1～2 day.
(4) utilize method culling pepper epidemic disease resistant breeding material of the present invention, required cost is low, only is 2 yuan/part～3 yuan of/part breeding materials.
Description of drawings
Fig. 1 utilizes method of the present invention to capsicum blight-resistant breeding material C M334 that generally acknowledges and the superseded result who feels epidemic disease resistant breeding material Early Calwonder.Among the figure, the 1st swimming lane is CM334, no 990bp band; The 2nd swimming lane is Early Calwonder, and the 990bp band is arranged; The 3rd swimming lane is dna molecular amount standard DL2000.
Fig. 2 is for utilizing method of the present invention antagonism eqpidemic disease pepper breeding material C M334, sense eqpidemic disease pepper breeding material Early Calwonder, and the qualification result of the pepper breeding material of 8 parts of anti-eqpidemic disease the unknowns such as A2, A11, B19, B27,2031,2056,2096, eggplant door.Wherein, the 1st swimming lane and the 2nd～4 swimming lane are respectively Early Calwonder and other 3 parts sense epidemic disease resistant breeding materials (B27, eggplant door and 2096), have the 990bp band to occur.The 5th swimming lane and the 6th～10 swimming lane are respectively CM334 and other 5 parts of blight-resistant breeding materials (A2, A11, B19,2056,2031), and no 990bp band occurs.The 11st swimming lane is dna molecular amount standard DL2000.
The present invention is described in further detail below in conjunction with accompanying drawing and application example.
Embodiment
The present invention is a method of utilizing the molecule marker culling pepper epidemic disease resistant breeding material, utilizes molecular marking technique that capsicum sense epidemic disease resistant breeding material is eliminated at the growth commitment first, and its concrete steps are as follows:
(1) will resist the breeding material of eqpidemic disease the unknown to be seeded in seedling pan and carry out Routine Management, temperature remains on 25 ℃～30 ℃, relative humidity 50%～70%, illumination 3000～4000Lx.When treating that seedling grows to 3～4 true leaves, with improvement SDS method (Gong Zhenhui etc., identify the minim DNA extracting method [J] of transfer-gen plant with PCR. Northwest Agricultural University's journal, 1997,25 (1): 45-47) extract the genomic dna of different breeding material young leaflet tablets respectively, measure DNA concentration with the nucleic acid-protein quantitative instrument, and DNA concentration is transferred to 25ng/ μ L with 1 * TE buffered soln.
(2) be template with above-mentioned genomic dna, behind the inoculation phytophthora that obtains according to the contriver in sense eqpidemic disease pepper breeding material forward and reverse primer of the gene order design molecule marker of special abduction delivering carry out pcr amplification reaction.Wherein, the nucleotides sequence of forward primer is classified as: 5 '-ctgtgatatggaaggggaacc-3 ', the nucleotides sequence of reverse primer is classified as: 5 '-aactcgctgggaaaagaatg-3 '.
The cumulative volume of pcr amplification reaction is 25 μ L, wherein contains 1 * amplification Buffer, 10ng～25ng template DNA, the MgCl of 2.0mmol/L
2, the forward primer of 0.4 μ mol/L, the reverse primer of 0.4 μ mol/L, the dNTPs of 0.2mmol/L, 0.5U Taq archaeal dna polymerase.Wherein, the response procedures of pcr amplification is: 95 ℃ of pre-sex change 5min, enter the PCR circulation again, and cycling program is 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 34 circulations altogether, extend 10min, 4 ℃ of preservations down at 72 ℃ at last.Pcr amplification reaction carries out on U.S. Bio-rad DNA cloning instrument.
(3) pcr amplification product carries out electrophoretic separation on 1.2% sepharose, is 1 μ g/mL ethidium bromide staining with final concentration, utilizes the Genesnap gel imaging system to observe, write down electrophoresis result.
According to the electrophoresis result that is write down, judge as follows:
If the feature band that it is 990bp that the electrophoresis result demonstration can amplify a length illustrates that this material is susceptible breeding material, it can be eliminated;
If the feature band that it is 990bp that the electrophoresis result demonstration can not amplify a length illustrates that this material is the breeding for disease resistance material, can be used for further capsicum blight-resistant breeding of new variety.
Below be concrete application example of the present invention, these all are more excellent examples, and application of the present invention is not limited to these embodiment.
Application Example 1:
(1) the capsicum sense epidemic disease resistant breeding material Early Calwonder that generally acknowledges and blight-resistant breeding material C M334 are seeded in seedling pan and carry out Routine Management, temperature remains on 25 ℃～30 ℃, relative humidity 50%～70%, illumination 3000Lx～4000Lx.When treating that seedling grows to 3～4 true leaves, with improvement SDS method (Gong Zhenhui etc., identify the minim DNA extracting method [J] of transfer-gen plant with PCR. Northwest Agricultural University's journal, 1997,25 (1): 45-47) extract the genomic dna of different parent's tender leafs respectively, measure DNA concentration with the nucleic acid-protein quantitative instrument, and DNA concentration is transferred to 25ng/ μ L with 1 * TE buffered soln.
(2) be template with above-mentioned genomic dna, behind the inoculation phytophthora that obtains according to the contriver in sense eqpidemic disease pepper breeding material the forward and reverse primer of the gene order design molecule marker of special abduction delivering carry out pcr amplification reaction.Wherein, the nucleotides sequence of forward primer is classified 5 '-ctgtgatatggaaggggaacc-3 ' as, and the nucleotides sequence of reverse primer is classified 5 '-aactcgctgggaaaagaatg-3 ' as.
The cumulative volume of pcr amplification reaction is 25 μ L, wherein contains 1 * amplification Buffer, 10ng～25ng template DNA, the MgCl of 2.0mmol/L
2, the forward primer of 0.4 μ mol/L, the reverse primer of 0.4 μ mol/L, the dNTPs of 0.2mmol/L, 0.5U Taq archaeal dna polymerase.
Wherein the program of pcr amplification reaction is: 95 ℃ of pre-sex change 5min, enter the PCR circulation again, and cycling program is 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 34 circulations altogether, extend 10min, 4 ℃ of preservations down at 72 ℃ at last.Pcr amplification reaction carries out on U.S. Bio-rad DNA cloning instrument.
(3) pcr amplification product carries out electrophoretic separation on 1.2% sepharose, is 1 μ g/mL ethidium bromide staining with final concentration, utilizes the Genesnap gel imaging system to observe, write down electrophoresis result.Show the feature band that it is 990bp that sense eqpidemic disease pepper breeding material Early Calwonder can amplify a length, and the anti-eqpidemic disease pepper breeding material C M334 feature band (referring to Fig. 1) that can not to amplify a length be 990bp according to electrophoresis result.
Application Example 2:
(1) the pepper breeding material of the capsicum of generally acknowledging being felt 8 parts of anti-eqpidemic disease the unknowns such as epidemic disease resistant breeding material Early Calwonder and blight-resistant breeding material C M334 and A2, A11, B19, B27,2031,2056,2096, eggplant door is pressed methods such as Luo Dexu (Luo Dexu etc., capsicum epidemic disease disease resistance Molecular Identification technical study [J]. northwest agricultural journal, 2008,17 (5): 76-80) carry out disease resistance and identify, promptly adopt root-pouring method to inoculate when plant 6～8 leaves, 1d irritates permeable before the inoculation.During inoculation, prick a hole at the about 3cm of distance seedling rhizome place with glass rod, hole depth 3cm, accurately drawing 3mL concentration is 1 * 10
4In/mL phytophthora zoospore suspension the filling orifice, every kind 10 strains repeat 3 times.Preserve moisture behind the 24h, at 28 ℃, relative humidity 90%, illumination is 4000Lx, impels morbidity in the growth cabinet of 12h.Inoculation back 7d investigation onset grade, and calculate disease index.
(2) the inoculation result shows that the disease index of Early Calwonder is 97.6, belongs to high sense epidemic disease resistant breeding material; The disease index of CM334 is 0, belongs to high blight-resistant breeding material.In the pepper breeding material of 8 parts of anti-eqpidemic disease the unknowns such as A2, A11, B19, B27,2031,2056,2096, eggplant door, show susceptible have 3 parts, be respectively B27, eggplant door and 2096, its disease index is respectively 80.7,72.7,54.2; Show disease-resistant have 5 parts, be respectively A11, A2, B19,2056,2031, its disease index is respectively 0 (high anti-), 5 (high anti-), 12.7 (disease-resistant), 42.5 (in anti-), 45.7 (in anti-).
(3) above-mentioned 10 parts of pepper breeding materials are seeded in seedling pan and carry out Routine Management, temperature remains on 25 ℃～30 ℃, relative humidity 50%～70%, illumination 3000Lx～4000Lx.When treating that seedling grows to 3～4 true leaves, with improvement SDS method (Gong Zhenhui etc., identify the minim DNA extracting method [J] of transfer-gen plant with PCR. Northwest Agricultural University's journal, 1997,25 (1): 45-47) extract the genomic dna of different parent's tender leafs respectively, measure DNA concentration with the nucleic acid-protein quantitative instrument, and DNA concentration is transferred to 25ng/ μ L with 1 * TE buffered soln.
(4) be template with above-mentioned genomic dna, behind the inoculation phytophthora that obtains according to the contriver in sense eqpidemic disease pepper breeding material the forward and reverse primer of the gene order design molecule marker of special abduction delivering carry out pcr amplification reaction.Wherein, the nucleotides sequence of forward primer is classified 5 '-ctgtgatatggaaggggaacc-3 ' as, and the nucleotides sequence of reverse primer is classified 5 '-aactcgctgggaaaagaatg-3 ' as.
The cumulative volume of pcr amplification reaction is 25 μ L, wherein contains 1 * amplification Buffer, 10ng～25ng template DNA, the MgCl of 2.0mmol/L
2, the forward primer of 0.4 μ mol/L, the reverse primer of 0.4 μ mol/L, the dNTPs of 0.2mmol/L, 0.5U Taq archaeal dna polymerase.The program of pcr amplification reaction is: 95 ℃ of pre-sex change 5min, enter the PCR circulation again, and cycling program is 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 34 circulations altogether, extend 10min, 4 ℃ of preservations down at 72 ℃ at last.Pcr amplification reaction carries out on U.S. Bio-rad DNA cloning instrument.
(5) pcr amplification product carries out electrophoretic separation on 1.2% sepharose, is 1 μ g/mL ethidium bromide staining with final concentration, utilizes the Genesnap gel imaging system to observe, write down electrophoresis result.
(6) electrophoresis result shows, the feature band that it is 990bp that sense eqpidemic disease pepper breeding material Early Calwonder and other 3 parts sense epidemic disease resistant breeding materials (B27,2096, eggplant door) can amplify a length, and anti-eqpidemic disease pepper breeding material C M334 and other 5 parts of blight-resistant breeding materials (A2, A11, B19,2056,2031) feature band (accompanying drawing 2) that can not to amplify a length be 990bp.
(7) the superseded result of molecule marker shows consistent with the superseded result of artificial inoculation in seedling stage.
Claims (3)
1, a kind of method of utilizing the molecule marker culling pepper epidemic disease resistant breeding material is characterized in that detailed process comprises the steps:
(1) will resist the pepper breeding material of eqpidemic disease the unknown to be seeded in seedling pan and carry out Routine Management, when treating that seedling grows to 3～4 true leaves, extract the genomic dna of different breeding material young leaflet tablets respectively with improvement SDS method;
(2) genomic dna with above-mentioned young leaflet tablet is a template, utilizes forward and reverse primer of molecule marker to carry out pcr amplification reaction;
The nucleotides sequence of the forward primer of molecule marker is classified as: 5 '-ctgtgatatggaaggggaacc-3 ', the nucleotides sequence of reverse primer is classified 5 '-aactcgctgggaaaagaatg-3 ' as
(3) pcr amplification product carries out electrophoretic separation on 1.2% sepharose, and ethidium bromide staining utilizes gel imaging system to observe, write down electrophoresis result;
(4) electrophoresis result is carried out following judgement:
If the feature band that it is 990bp that the electrophoresis result demonstration can amplify a length illustrates this breeding material for sense eqpidemic disease pepper breeding material, and it is superseded;
If the feature band that it is 990bp that the electrophoresis result demonstration can not amplify a length illustrates that this breeding material is anti-eqpidemic disease pepper breeding material.
2, the method for claim 1 is characterized in that, the cumulative volume of pcr amplification reaction is 25 μ L in the described step (2), wherein contains 1 * amplification Buffer, 10ng～25ng template DNA, 2.0mmol/L MgCl
2, the forward primer of 0.4 μ mol/L, the reverse primer of 0.4 μ mol/L, 0.2mmol/LdNTPs, 0.5U Taq archaeal dna polymerase.
3, the method for claim 1, it is characterized in that, the program of pcr amplification reaction is in the described step (2): 95 ℃ of pre-sex change 5min, enter the PCR circulation again, cycling program is 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, carry out 34 circulations altogether, extend 10min, 4 ℃ of preservations down at 72 ℃ at last.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878093A (en) * | 2015-04-30 | 2015-09-02 | 中国农业大学 | Molecular marker in close linkage with chilli blight resistance gene and application of molecular marker |
| CN113785741A (en) * | 2021-11-18 | 2021-12-14 | 山东寿光蔬菜种业集团有限公司 | Seed production method for pepper breeding |
-
2009
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878093A (en) * | 2015-04-30 | 2015-09-02 | 中国农业大学 | Molecular marker in close linkage with chilli blight resistance gene and application of molecular marker |
| CN104878093B (en) * | 2015-04-30 | 2017-06-16 | 中国农业大学 | A kind of molecular labeling and its application with capsicum blight-resistant gene close linkage |
| CN113785741A (en) * | 2021-11-18 | 2021-12-14 | 山东寿光蔬菜种业集团有限公司 | Seed production method for pepper breeding |
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| CN101487049B (en) | 2011-04-06 |
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