CN102312000A - Method of detecting double rice blast resistance genes Pi1 and Pi2 by using molecular marker-assisted selection - Google Patents
Method of detecting double rice blast resistance genes Pi1 and Pi2 by using molecular marker-assisted selection Download PDFInfo
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Abstract
The invention discloses a method of detecting double rice blast resistance genes Pi1 and Pi2 by using molecular marker-assisted selection. According to the method, by using DNA extracted from a plant and two pairs of SSR marks, i.e., P1 and P2, which are respectively in close linkage with rice blast resistance genes Pi1 and Pi2, PCR amplification and polyacrylamide gel electrophoresis separation are carried out on donor parent and acceptor parent of the rice blast resistance genes and genome DNA of hybridized or backcrossed generation of the donor parent and the acceptor parent, synchronized detection of genotype of each individual plant at two rice blast resistance gene loci is carried out, and individual plants which have introduced the rice blast resistance genes are selected from the hybridized or backcrossed generation through comparison of genotype so as to provide guidance to hybridization matching and screening of target materials in the next step of breeding process. The method provided in the invention has substantially improved detection efficiency compared to a normal detection method of extracting DNA by the CTAB method and sequentially detecting genotype of a single plant at the two rice blast resistance gene loci with a single mark; cost for the method is greatly reduced as well; the method meets demands of scale and industrialized molecular marker-assisted breeding.
Description
Technical field
The invention belongs to the molecular breeding technical field, relate to the dual-gene detection method of molecular marker assisted selection, the detection method of the dual-gene Pi1 of particularly a kind of molecular marker assisted selection blast resisting, Pi2.
Background technology
Rice blast is to influence one of important disease of Rice Production in the worldwide; Existing more than 80 country's these diseases of report in the whole world take place; Nearly 1,000 ten thousand tons of the grain loss that causes every year; The paddy loss that China causes every year thus also reaches more than one hundred million kilograms, and cultivating and plant disease-resistant variety is the most effective and safest measure of control rice blast.
But traditional breeding for disease resistance needs a large amount of field work; Cycle is long; Simultaneously the selection of breeding objective is received the interference of several factors, and the kind of newly breeding is after continuous multi-generation plantation, the resistance of kind is because of the physiological strain variation loss very soon of Pyricularia oryzae; The blast resisting ability often shows decline, thereby cultivates the important topic that the new rice variety with durable resistance becomes the paddy rice scientific research.And the molecular marker assisted selection that develops rapidly in recent years technology (MAS) can remedy traditional breeding method and selects the low shortcoming of technological accuracy rate to accelerate breeding process; Select anti-spectrum disease-resistant gene wide, the tool durable resistance to cultivate new variety simultaneously again, thereby prolong the life-span of blast resisting kind as anti-source.So far there be more than 80 blast resistant gene to be labeled the location in the paddy rice, wherein cloned for 13.Certified resistance of wide spectrum gene comprises: Pi1, Pi2, Pi3, Piz, Pi9, Piz-t, Pi33, Pigm etc.In recent years, disease-resistant gene has more application in the paddy disease-resistant molecular breeding, Hittalmani etc. with Pi1, Piz5 and Pita gene pyramiding in rice strain BL125; Narayanan etc. are aggregated to Pi1, Piz5 among the CO39; It is medium that Chen Xuewei etc. are aggregated to G46B with Pid1, Pib and Pita.Facts have proved, utilize the molecular marker assisted selection technology, different blast resistant genes is aggregated to the lasting anti-pest property that can effectively strengthen rice varieties in the kind.
Molecular marker assisted selection technology (MAS) is though be applied to breeding practice, but still one of problem that needs to solve is the detection cost that reduces the marker gene type, thereby can make it, mass-producing apply to go in the business-like procedure of breeding on a large scale.Yet, conventional DNA detection program, the leaching process of its DNA is complicated, wastes time and energy; Big and the long reaction time of the amount of reagent that the PCR reaction system expends; Can not carry out synchronous detection to a plurality of marker sites in the same individuality.Therefore, as far as big breeding population, its marker gene type detects length consuming time, and cost is also higher relatively.
The object of the present invention is to provide a kind of in rice anti-rice blast molecular mark process; Can carry out fast dual-gene Pi1 of blast resisting and Pi2, accurately, the method for stable detection, with the needs of the molecular mark that is applicable to mass-producing, industrialization.
Its technical scheme is: the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting, Pi2 is characterized in that its concrete steps are:
1) DNA extraction:
Get about single-strain blade 1.0cm * 1.0cm the sample of size, put into 96 hole PCR pipe, add NaOH solution, add Tris-HCI after in boiling water, boiling 45sec, it is for use in boiling water, to boil the rearmounted cooled on ice of 2min;
2) PCR reaction:
In the PCR reaction tubes, add sample DNA extracting solution, Buffer, dNTP, primer P1, primer P2, ddH respectively
2O and Taq enzyme; 94 ℃ of preparatory sex change 4 of elder generation '; 94 ℃ of sex change 15 again ", 55 ℃ of annealing 15 ", 72 ℃ are extended 30 " and, 35 circulations; 72 ℃ extend 7 '; After reaction is accomplished, add 6 * Loading buffer termination reaction, it is subsequent use to put 4 ℃ of environment under preservation;
3) polyacrylamide gel electrophoresis:
Separation gel is slowly injected between two sheet glass, leave standstill and solidify more than half a hour; Sheet glass is loaded onto electrophoresis chamber, 2000V, 90W preheating half a hour; Insert comb, point sample; 2000V, electrophoresis under the 85W condition; Behind the following glue, with offset plate with clear water rinsing 15 "; Put into the staining fluid basin and dye 8 '; Take out offset plate, with clear water rinsing 25 "; Put into developing solution and develop and be with clearly to DNA, wash 1 again '; Treat that offset plate dries back observed and recorded result under the lamp box of reading the X-ray sheet;
4) interpretation of result:
Obtain the electrophoretic band of blast resistant gene donor parents, receptor parent and both hybridization or backcross progeny respectively with aforesaid method; Contrast range gene type spectrum band; Find out the situation of the concrete Pi1 of importing and two blast resistant gene individual plants of Pi2 in hybridization or the backcross progeny colony, so that instruct next step hybridization apolegamy and the screening of purpose material in the procedure of breeding.
Its technique effect is: the present invention adopts the rapid DNA extraction method to extract the DNA of plant; Utilizing two couples of SSR mark: P1 (with blast resistant gene Pi1 close linkage) and P2 (with blast resistant gene Pi2 close linkage) that the genomic dna of donor parents (containing Pi1 and Pi2 blast resistant gene simultaneously), receptor parent (not containing Pi1 or Pi2 blast resistant gene) and both hybridization or the backcross progeny of blast resistant gene is carried out pcr amplification separates with polyacrylamide gel electrophoresis; The genotype of each individual plant of synchronous detection on two disease-resistant gene sites; Comparison through the range gene type; Find out and imported single in hybridization or the backcross progeny colony or imported the individual plant of two blast resistant genes simultaneously, instruct next step hybridization apolegamy and the screening of purpose material in the procedure of breeding.The conventional CTAB method of its detection efficiency is extracted DNA; And single mark detects the genotype of individual plant in two disease-resistant sites one by one significant raising has been arranged; Simultaneously cost has also obtained reducing largely, is applicable to the molecular mark (for details see attached table 1) of mass-producing, industrialization.
Description of drawings
Fig. 1 be in the rice anti-rice blast molecular mark process backcross population part individual plant at the genotype bands of a spectrum of two marker sites.
Embodiment
The detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting, Pi2, its concrete steps are following:
1) DNA extraction:
Get the sample of size about single-strain blade 1.0cm * 1.0cm; Put into 96 hole PCR pipe; Add the NaOH solution of 40 μ L 0.30mol/L, in boiling water, boil 45sec, add the Tris-HCI (pH 3) of 40 μ L 0.17mol/L then; In boiling water, boil the rearmounted cooled on ice of 2min, get the 1ul extracting solution and supply pcr amplification reaction or 4 ℃ of preservations down.
2) PCR reaction:
(1) reaction system (10ul): in the PCR pipe, add the primer P1 of 1ul DNA extraction liquid, 1ulBuffer, 0.4ul dNTP, 0.25ul respectively, the primer P2 of 0.25ul, the Taq enzyme of 0.1ul and the ddH of 7ul
2O.
Primer P1 sequence is P1F:5 '-GTGCATGAGTCCAGCTCGCA-3 ', P1R:5 '-GTGTACTCCCATGGCTGCTC-3 ';
Primer P2 sequence is P2F:5 '-ATTGCTGCAAAGTGGGAGAC-3 ', P2R:5 '-AAGTGGAGGCAGTTCACCAC-3 '.
(2) response procedures:
94 ℃ of preparatory sex change 4 of elder generation ';
94 ℃ of sex change 15 again ", 55 ℃ of annealing 15 ", 72 ℃ are extended 30 " and, 35 circulations;
The 72 ℃ of extensions 7 in back '.
After reaction is accomplished, add 6 * Loading buffer termination reaction, it is subsequent use to put 4 ℃ of environment under preservation.
3) polyacrylamide gel electrophoresis
1. glass cleaning plate: firmly clean with 95% alcohol and to be coated with that two panels of peeling off silane and affine silane.
2. be coated with affine silane: the mixed solution of affine liquid of 1ml and the affine silane of 18ul is applied on the sheet glass that does not have groove equably.
3. be coated with and peel off silane: smear 1ml on the reeded sheet glass equably and peel off silane.
4. add edge strip, dress plate: will not have the sheet glass (scribbling one of affine silane faces up) of groove to lie against on the experiment table, edge strip is placed in both sides alignment, and with reeded sheet glass (scribble peel off one of silane face down) lid on it, the zygomorphy folder is gone up clip.
5. irritate separation gel: separation gel is slowly injected between two sheet glass, oppositely plug comb, leave standstill and solidify more than half a hour.Separation gel is that 4% SEPIGEL 305,400ul concentration are that 10% ammonium persulphate APS and the N,N,N TEMED of 40ul mix by 65ml concentration.
6. electrophoresis: pull up comb, sheet glass is loaded onto electrophoresis chamber, 2000V, 90W preheating half a hour; Forward is plugged comb, point sample; 2000V, electrophoresis under the 85W condition, every electrophoresis repeated point sample once after 15 minutes, repeated point sample 2～4 times, and the sample of each point sample is different.
7. dye and develop: behind the following glue, with offset plate with clear water rinsing 15 "; Put into staining fluid (AgNO32L of 1g/L) basin and dye 8 '; Take out offset plate, with clear water rinsing 25 "; Putting into develops in the developing solution (the NaOH 2L of 20g/L, add 10ml formaldehyde) be with to DNA clear; Wash 1 again '.
8. observe: offset plate dries back observed and recorded result under the lamp box of reading the X-ray sheet.
4) interpretation of result:
Obtain respectively with aforesaid method: blast resistant gene donor parents (containing Pi1 and Pi2 blast resistant gene simultaneously), receptor parent (not containing Pi1 or Pi2 blast resistant gene) and both hybridization or the electrophoretic band of backcross progeny; Contrast range gene type spectrum band; Find out the concrete Pi1 of importing and two blast resistant gene individual plants of Pi2 in hybridization or the backcross progeny colony, so that instruct next step hybridization apolegamy and the screening of purpose material in the procedure of breeding.As shown in Figure 1, number " 1 " is represented the acceptor gene type among the figure, and the heterozygous genes type is represented in " 2 ".Wherein individual plant exists, and when 1. the genotype of P1, two marker sites of P2 was " 2 ", representative had been imported Pi1 and two blast resistant genes of Pi2 simultaneously; When 2. the genotype of a marker site was " 2 ", representative only had been imported into a blast resistant gene among Pi1 or the Pi2; When 3. two marker site genotype were " 1 ", then representative was not imported into Pi1 and Pi2 blast resistant gene.Simultaneously; By the electrophoretic band of the visible P1 amplified production of Fig. 1 electrophoretic band downstream at the P2 amplified production: the DNA cloning fragment length of P1 in acceptor is shorter than its expanding fragment length in donor, and the electrophoretic band that on running gel figure, shows as acceptor is positioned at the electrophoretic band downstream of donor; And the DNA cloning fragment length of P2 in acceptor is longer than the expanding fragment length in its donor, and the electrophoretic band that on running gel figure, shows as acceptor equally is positioned at the electrophoretic band downstream of donor.
Claims (7)
1. the detection method of the dual-gene Pi1 of molecular marker assisted selection blast resisting, Pi2 is characterized in that its concrete steps are:
1) DNA extraction:
Get about single-strain blade 1.0cm * 1.0cm the sample of size, put into 96 hole PCR pipe, add NaOH solution, add Tris-HCI after in boiling water, boiling 45sec, it is for use in boiling water, to boil the rearmounted cooled on ice of 2min;
2) PCR reaction:
In the PCR reaction tubes, add sample DNA extracting solution, Buffer, dNTP, primer P1, primer P2, ddH respectively
2O and Taq enzyme; 94 ℃ of preparatory sex change 4 of elder generation '; 94 ℃ of sex change 15 again ", 55 ℃ of annealing 15 ", 72 ℃ are extended 30 " and, 35 circulations; 72 ℃ extend 7 '; After reaction is accomplished, add 6 * Loading buffer termination reaction, it is subsequent use to put 4 ℃ of environment under preservation;
3) polyacrylamide gel electrophoresis:
Separation gel is slowly injected between two sheet glass, leave standstill and solidify more than half a hour; Sheet glass is loaded onto electrophoresis chamber, 2000V, 90W preheating half a hour; Insert comb, point sample; 2000V, electrophoresis under the 85W condition; Behind the following glue, with offset plate with clear water rinsing 15 "; Put into the staining fluid basin and dye 8 '; Take out offset plate, with clear water rinsing 25 "; Put into developing solution and develop and be with clearly to DNA, wash 1 again '; Treat that offset plate dries back observed and recorded result under the lamp box of reading the X-ray sheet;
4) interpretation of result:
Obtain the electrophoretic band of blast resistant gene donor parents, receptor parent and both hybridization or backcross progeny respectively with aforesaid method; Contrast range gene type spectrum band; Find out the situation of the concrete Pi1 of importing and two blast resistant gene individual plants of Pi2 in hybridization or the backcross progeny colony, so that instruct next step hybridization apolegamy and the screening of purpose material in the procedure of breeding.
2. the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting according to claim 1, Pi2, it is characterized in that: the volume ratio of NaOH solution and Tris-HCI solution is 1: 1 in the said sample DNA extracting solution.
3. the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting according to claim 1, Pi2; It is characterized in that: P1 and two pairs of primers of P2 of adding in the said PCR reaction; Wherein, Primer P1 sequence is P1F:5 '-GTGCATGAGTCCAGCTCGCA-3 ', P1R: 5 '-GTGTACTCCCATGGCTGCTC-3 '; Primer P2 sequence is P2F:5 '-ATTGCTGCAAAGTGGGAGAC-3 ', P2R5 '-AAGTGGAGGCAGTTCACCAC-3 '.
4. the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting according to claim 1, Pi2 is characterized in that: sample DNA extracting solution, Buffer, dNTP, primer P1, primer P2, Taq enzyme and ddH in the said PCR reaction
2The volume ratio of O is: 1.0: 1.0: 0.4: 0.25: 0.25: 0.1: 7.0.
5. the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting according to claim 1, Pi2 is characterized in that: the program of said PCR reaction be 94 ℃ of preparatory sex change 4 earlier '; 94 ℃ of sex change 15 again ", 55 ℃ of annealing 15 ", 72 ℃ are extended 30 " and, 35 circulations; 72 ℃ extend 7 '.
6. the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting according to claim 1, Pi2; It is characterized in that: said separation gel is by the SEPIGEL 305 of concentration 4%, the ammonium persulphate and the N of concentration 10%; N; N ', N '-Tetramethyl Ethylene Diamine mixes, and its mixed volume ratio is 65: 0.4: 0.04.
7. the detection method of the dual-gene Pi1 of a kind of molecular marker assisted selection blast resisting according to claim 1, Pi2; It is characterized in that: said polyacrylamide gel electrophoresis detects; Be at 2000V, under the 85W condition, every electrophoresis repeated point sample once after 15 minutes; Repeat point sample 2～4 times, and the sample of each point sample is different.
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Cited By (13)
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| CN102703443A (en) * | 2012-05-23 | 2012-10-03 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN103215369A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103215368A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2 |
| CN103215370A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting rice blast-resistant gene pi5 |
| CN103320437A (en) * | 2013-07-10 | 2013-09-25 | 广东省农业科学院植物保护研究所 | Gene-specific molecular marker Pi2SNP of rice blast-resistant gene Pi2 as well as preparation method and application thereof |
| CN104087578A (en) * | 2014-07-07 | 2014-10-08 | 合肥丰乐种业股份有限公司 | Molecular marker closely linked with rice bacterial blight resistance gene and primers and application thereof |
| CN104762393A (en) * | 2015-04-09 | 2015-07-08 | 湖北文理学院 | Method for improving amplification and detection efficiency of SSR molecular marker |
| CN105779622A (en) * | 2016-04-21 | 2016-07-20 | 南京农业大学 | Molecular marker closely linked with race-specific rice-blast-resistant gene of paddy rice and application of molecular marker |
| CN106480049A (en) * | 2015-08-24 | 2017-03-08 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010212 and its detection method |
| CN107304449A (en) * | 2016-04-22 | 2017-10-31 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010311 and its detection primer and application |
| CN107304427A (en) * | 2016-04-22 | 2017-10-31 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010005 and its detection primer and application |
| CN108901831A (en) * | 2018-08-17 | 2018-11-30 | 江苏里下河地区农业科学研究所 | The selection of the strong advantage Indica Rice Restorer Lines of blast resisting |
| CN109385466A (en) * | 2018-12-28 | 2019-02-26 | 广东省农业科学院水稻研究所 | The KASP Functional marker of resistance gene of rice blast Pi2 a kind of and its application |
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| CN102703443B (en) * | 2012-05-23 | 2014-09-24 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN102703443A (en) * | 2012-05-23 | 2012-10-03 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN103215369A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103215368A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2 |
| CN103215370A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting rice blast-resistant gene pi5 |
| CN103215369B (en) * | 2013-05-08 | 2014-07-23 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103320437B (en) * | 2013-07-10 | 2015-04-29 | 广东省农业科学院植物保护研究所 | Gene-specific molecular marker Pi2SNP of rice blast-resistant gene Pi2 as well as preparation method and application thereof |
| CN103320437A (en) * | 2013-07-10 | 2013-09-25 | 广东省农业科学院植物保护研究所 | Gene-specific molecular marker Pi2SNP of rice blast-resistant gene Pi2 as well as preparation method and application thereof |
| CN104087578A (en) * | 2014-07-07 | 2014-10-08 | 合肥丰乐种业股份有限公司 | Molecular marker closely linked with rice bacterial blight resistance gene and primers and application thereof |
| CN104762393A (en) * | 2015-04-09 | 2015-07-08 | 湖北文理学院 | Method for improving amplification and detection efficiency of SSR molecular marker |
| CN106480049A (en) * | 2015-08-24 | 2017-03-08 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010212 and its detection method |
| CN106480049B (en) * | 2015-08-24 | 2020-06-19 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010212 and detection method thereof |
| CN105779622A (en) * | 2016-04-21 | 2016-07-20 | 南京农业大学 | Molecular marker closely linked with race-specific rice-blast-resistant gene of paddy rice and application of molecular marker |
| CN107304449A (en) * | 2016-04-22 | 2017-10-31 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010311 and its detection primer and application |
| CN107304427A (en) * | 2016-04-22 | 2017-10-31 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010005 and its detection primer and application |
| CN107304449B (en) * | 2016-04-22 | 2021-12-14 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010311 and detection primer and application thereof |
| CN107304427B (en) * | 2016-04-22 | 2021-12-14 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR010005 as well as detection primer and application thereof |
| CN108901831A (en) * | 2018-08-17 | 2018-11-30 | 江苏里下河地区农业科学研究所 | The selection of the strong advantage Indica Rice Restorer Lines of blast resisting |
| CN109385466A (en) * | 2018-12-28 | 2019-02-26 | 广东省农业科学院水稻研究所 | The KASP Functional marker of resistance gene of rice blast Pi2 a kind of and its application |
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