CN102373291B - RAPD-SCAR839 label for identifying group E chromosome of thinopyrum elongatum and application of same - Google Patents
RAPD-SCAR839 label for identifying group E chromosome of thinopyrum elongatum and application of same Download PDFInfo
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Abstract
The invention discloses an RAPD-SCAR83 label for identifying group E chromosome of thinopyrum elongatum and application of the same. A nucleotide sequence of the group E chromosome is as shown in SEQ. ID. NO. 2 or SEQ. ID. NO. 5. In the invention, specific RAPD segments are screened to obtain through an RAPD-SCAR method, and are converted into stable SCAR labels; and amplification of the RAPD labels is detected. When the label of the group E chromosome of the thinopyrum elongatum provided by the invention is adopted, a solid foundation is laid for quickly identifying a 2E or 3E chromatin or chromosome of the thinopyrum elongatum under the genetical background of wheat through the detection of the SCAR labels, and a very convenient and quick means is also provided for molecular marker-assisted breeding.
Description
Technical field
The invention belongs to the technical field that long fringe couchgrass E group chromosome is identified, relate to a kind of RAPD-SCAR that identifies long fringe couchgrass E group chromosome
839Mark and application thereof.
Background technology
Wheat is one of important in the world food crop.It is the second largest farm crop that are only second to paddy rice in China, and Wheat Production has very important strategic importance to the national economic development.Because the Wheat cultivar simplification makes its hereditary basis day by day narrow, anti-source is deficient, has greatly limited the further improvement of its yield and quality.Existing a large amount of heritable variation in the nearly edge species of wheat, is that wheat is improved utilizable desirable genes resource, and it is geneticist and the unfailing research topic of breeding scholar that these desirable genes are imported wheat always.Genus Agropyron is that a sibling species of wheat belongs to, it has many good characters that common wheat lacks, strong such as tillering ability, well developed root system, cold-resistant, drought resisting, barren-resistant, Salt And Alkali Tolerance power is strong, spends more, large fringe, how real, the baking properties that grain protein content is high and good, anti-multiple diseases ability is strong, to immunity, is one of important parent of wheat genetic breeding to stripe rust of wheat, leaf rust, black rust, Powdery Mildew, yellow dwart, stripe mosaic disease and bunt high resistance.Long fringe couchgrass (Agropyron elongatum) is an important sibling species of wheat, having high, the anti-multiple fungi of protein content and virus disease, drought-enduring, cold-resistant, large fringe and the excellent proterties such as spend more, is one of most widely used wild species in wheat breed is improved.
At nature, long fringe couchgrass has diploid, tetraploid and 3 kinds of ploidy types of decaploid.Wherein Diploid Thinopyron elongatum grass (Thinopyrum elongatum) E group chromosome is the basic genome that forms Genus Agropyron (Elytrigia) polyploid species, carry the gene useful to the wheat genetic breeding, but the genome for tetraploid and decaploid type consists of, and never has consistent conclusion.It is a fruitful approach that method by distant hybirdization and chromosome engineering belongs to from the common wheat wild relatives that the method that shifts excellent genes has been proved.At present, Sr24 (3DL), Sr26 (6AL), Sr25 (7DL, 7AL), Lr19, the genes such as Lr24 from long fringe couchgrass, have successfully been shifted abroad.Domesticly little lay down No. 4, No. 5, No. 6, the little wheat breeds such as 107 of laying down from wheat and long fringe couchgrass hybrid generation, have been bred.
A kind of effective means that good character and excellent gene with exogenous chromosome import wheat by hybridization and the mode of gradually oozing, but, in the wheat of restructuring, the external source E group chromatin or the chromosomal detection that come from wheat-long fringe couchgrass filial generation become vital problem, although by the key property on the agronomy and biochemical analysis with valuable genetic mapping on long fringe couchgrass karyomit(e), but the detection means of these experiments is unsettled, and is not suitable for carrying out the detection of important agronomy character.Some genetics means are arranged, such as technology such as Chromosomes Banding, in situ hybridizations, used it for the detection of exogenous genetic material under the wheat genetic background, but these technology are time-consuming, effort, requirement to technology is also very high, and carries out large-scale offspring's rapid screening with these laboratory facilities and still have certain limitation.So an urgent demand exploitation is a kind of can identify long fringe couchgrass external source chromatin or chromosomal molecular marking technique fast, accurately, on a large scale under the wheat genetic background.
Along with molecular biological development, dna molecular marker has been widely used in the research of wheat genetic breeding, restrictive fragment length polymerphism (RFLP) is the molecule marker that is used first, in plant genetic research, obtain very large using value, but, the DNA of RFLP Technology Need large-scale purification, and experiment itself is time-consuming, also very high to technical requirements, it is not suitable for carrying out the large-scale screening of breeding system, and therefore, the breeding scholar has begun to carry out the research of regular-PCR, be desirably in and find a kind of simpler mode to identify external source chromatin in the wheat breeding system, the dna molecular marker technology of many PCR-based is developed, such as RAPD, and AFLP, EST, SSR etc.In these marks, RAPD (Random Amplified Polymorphic DNA) technology is relatively simple, and it does not need to know the information of relevant sequence aspect, and the application in finger printing and phylogenetics research field is efficient simultaneously.But its repeatability and stability are very poor, if be translated into the SCAR mark, its specificity and stability increase substantially, and can be applied to more convenient and quicker the detection of chromosome nonhomologous.
Summary of the invention
The problem that the present invention solves is to provide a kind of RAPD-SCAR that identifies long fringe couchgrass E group chromosome
839Mark and application thereof lay the foundation for identifying quickly and accurately the long fringe couchgrass of external source E group chromatin or karyomit(e) under the wheat genetic background.
The present invention is achieved through the following technical solutions:
The long fringe couchgrass of a kind of evaluation E group chromosome specific mark gene, its nucleotide sequence is shown in SEQ.ID.NO.2 or SEQ.ID.NO.5.
A kind of primer of the mark shown in the SEQ.ID.NO.2 that increases, the nucleotide sequence of its primer is shown in SEQ.ID.NO.1.
A kind of primer pair of the mark shown in the SEQ.ID.NO.5 that increases, the nucleotide sequence of its upstream and downstream primer are respectively shown in SEQ.ID.NO.3, SEQ.ID.NO.4.
Sequence shown in the SEQ.ID.NO.5 is as long fringe couchgrass 2E or 3E chromosomal evaluation or the tracking detection of SCAR tag application under the wheat genetic background.
Described application is based on the molecular mark of long fringe couchgrass 2E or 3E karyomit(e) system cultivation wheat breed.
Described application is to carry out pcr amplification with the upstream and downstream primer pair testing gene group shown in SEQ.ID.NO.3, the SEQ.ID.NO.4, then according to the electrophoresis result of amplified production, to whether containing long fringe couchgrass 2E or 3E karyomit(e) is judged.
The amplification program of described pcr amplification is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 60s, 30～35 circulations; 72 ℃ are extended 10min.
If contain the target fragment of 839bp in the middle of the electrophoresis result of amplified production, then contain long fringe couchgrass 2E or 3E karyomit(e) in the testing gene group; If there is not the target fragment of 839bp in the middle of the electrophoresis result of amplified production, then not long fringe couchgrass 2E or 3E karyomit(e) in the testing gene group.
Compared with prior art, the present invention has following useful technique effect:
1, the present invention adopts the RAPD molecular marking technique to the analysis of the polymorphism of " China spring " wheat, Diploid Thinopyron elongatum grass, three kinds of long fringe couchgrass of material of Tetraploid Elytrigia E group chromosome, filter out the RAPD specific fragment of long fringe couchgrass E group chromosome, and then be converted into stable SCAR mark; Such SCAR mark has had the specificity of long fringe couchgrass E group chromosome, has improved the accuracy that detects, and is very beneficial for the evaluation of the long fringe couchgrass E group chromosome under the wheat genetic background and follows the tracks of detection.Compare with existing authentication method, utilize this SCAR mark to identify that then accuracy will obviously promote undoubtedly, and be very convenient, fast.
2, the SCAR mark of the present invention's acquisition is called SCAR
839Utilize common wheat " China spring ", " China spring "-gametocidal chromosome 2C disomic addition line, Diploid Thinopyron elongatum grass, Tetraploid Elytrigia material to carry out the checking of SCAR mark, in Diploid Thinopyron elongatum grass, Tetraploid Elytrigia, amplified the fragment of 839bp, and in common wheat " China spring ", " China spring "-gametocidal chromosome 2C disomic addition line this product not, this mark of preliminary proof can be used for the detection of long fringe couchgrass E group chromosome.
Further use this to the hybrid F of SCAR primer pair " China spring "-gametocidal chromosome 2C disomic addition line with " China spring "-long fringe couchgrass 3E disomic addition line
1Generation, F
2In generation, analyzed, and the result is at whole F
1Generation and part F
2For having amplified the purpose fragment in the material, further proved this SCAR
839Mark can be used for detecting the long fringe couchgrass E group chromosome under the Wheat Background, provides new way for the quick tracking of long fringe couchgrass E group chromosome in the Wheat Background detects.
3, the detection of used SCAR mark, the PCR reaction system of use does not need special PCR reaction kit and special reaction reagent, requires the same with other common PCR reaction; Only need to after pcr amplification, pass through electrophoresis detection, just can have or not the identical purpose band of size to judge according to electrophoresis result, simple to operation.
4, the mark of long fringe couchgrass E group chromosome provided by the invention, detection by the SCAR mark, have laid a good foundation for long fringe couchgrass E group external source chromatin or karyomit(e) under the Rapid identification wheat genetic background, also provide very convenient efficiently approach for molecular mark simultaneously.
Description of drawings
Fig. 1 is the amplification of RAPD primer LW10 in three kinds of differing materials, and swimming lane 1 is common wheat " China spring ", and swimming lane 2 is the Diploid Thinopyron elongatum grass, and swimming lane 3 is Tetraploid Elytrigia (Marker:DL2000);
Fig. 2 is nucleotide sequence SEQ.ID No.2 (being called LW10), carries out the result of Blast search in NCBI;
Fig. 3 is SCAR
839Be marked at four kinds of amplifications in the differing materials, swimming lane 1 is " China spring ", swimming lane 2 is the Diploid Thinopyron elongatum grass, and swimming lane 3 is Tetraploid Elytrigia, and swimming lane 4 is " China spring "-gametocidal chromosome 2C disomic addition line (Marker:DL2000);
Fig. 4 is SCAR
839The positioning result of molecule marker in " China spring "-long fringe couchgrass 1E～7E one cover disomic addition line, swimming lane 1 is " China spring "-long fringe couchgrass 1E disomic addition line, swimming lane 2 is " China spring "-long fringe couchgrass 2E disomic addition line, swimming lane 3 is " China spring "-long fringe couchgrass 3E disomic addition line, swimming lane 4 is " China spring "-long fringe couchgrass 4E disomic addition line, swimming lane 5 is " China spring " " China spring "-long fringe couchgrass 5E disomic addition line, swimming lane 6 is " China spring "-long fringe couchgrass 6E disomic addition line, swimming lane 7 is " China spring "-long fringe couchgrass 7E disomic addition line, swimming lane 8 is " China spring ", swimming lane 9 is the Diploid Thinopyron elongatum grass, swimming lane 10 is Tetraploid Elytrigia, and swimming lane 11 is " China spring "-gametocidal chromosome 2C disomic addition line (Marker:DL2000);
Fig. 5 is SCAR
839Be marked at " China spring "-long fringe couchgrass 3E disomic addition line and " China spring "-gametocidal chromosome 2C disomic addition line hybrid generation F
1, F
2In amplification, swimming lane 1～9 F that to be " China spring "-long fringe couchgrass 3E disomic addition line obtain with the hybridization of " China spring "-gametocidal chromosome 2C disomic addition line
1For material, swimming lane 10～19 is F
1F for the selfing acquisition
2For material, swimming lane 20 is common wheat-" China spring ", and swimming lane 21 is " China spring "-gametocidal chromosome 2C disomic addition line, and swimming lane 22 is the Diploid Thinopyron elongatum grass, and swimming lane 23 is Tetraploid Elytrigia (Marker:DL2000).
Embodiment
The invention provides a kind of RAPD-SCAR that identifies long fringe couchgrass E group chromosome
839Mark and application thereof obtain specific RAPD fragment by the screening of RAPD-SCAR method, and are converted to stable SCAR mark, and its amplification is detected.Be described in further detail below in conjunction with preparation, amplification and the evaluation of specific embodiment to mark, the explanation of the invention is not limited.
Related material source:
" China spring " wheat: derive from Harbin Teachers' Univ.'s molecular cytogenetics and genetic breeding key lab, in Harbin Teachers' Univ.'s molecular cytogenetics and base, genetic breeding major test nursery, extract the material kind DNA and carry out the molecular marker screening test in April, 2010 after 2 months.
Diploid Thinopyron elongatum grass: derive from Japanese national Biological resources plan wheat center (http://www.shigen.nig.ac.jp/-wheat/komugi/top.jsp), in January, 2010 with the material kind in Harbin Teachers' Univ.'s molecular cytogenetics and genetic breeding greenhouse, extract DNA after 3 months, save backup.
Tetraploid Elytrigia: derive from the Chinese Academy of Agricultural Sciences, in Harbin Teachers' Univ.'s molecular cytogenetics and genetic breeding greenhouse, extract DNA after 3 months with the material kind in January, 2010, saves backup.
" China spring "-gametocidal chromosome 2C disomic addition line: derive from Japanese national Biological resources plan wheat center (http://www.shigen.nig.ac.jp/-wheat/komugi/top.jsp), in Harbin Teachers' Univ.'s molecular cytogenetics and base, genetic breeding major test nursery, extract the material kind DNA and carry out the molecular marker screening test in April, 2010 after 2 months.
1, RAPD molecule marking method screening specificity RAPD fragment
Utilize the RAPD molecule marking method, in common wheat " China spring ", Diploid Thinopyron elongatum grass, Tetraploid Elytrigia, carry out the screening of polymorphism, obtain specific fragment.
1.1 adopt the CTAB method from fresh differing materials young leaflet tablet, to extract respectively genomic dna, and detect DNA concentration and quality;
1.2RAPD fragment amplification
The PCR reaction system: dna profiling 30ng, 10 * PCR buffer, 2.0 μ l, dNTP Mix (2.5mM) 1.5 μ l, RAPD primer (10 μ M) 1 μ l, r Taq polymerase (5U/ μ l) 0.2 μ l, the sterilization ultrapure water complements to 20 μ l;
The PCR response procedures:
In the process of screening, utilize common wheat " China spring ", Diploid Thinopyron elongatum grass, three kinds of materials of Tetraploid Elytrigia that 36 RAPD primers are screened, the foundation of screening is to have polymorphism in common wheat " China spring " and Diploid Thinopyron elongatum grass, Tetraploid Elytrigia bi-material, Diploid Thinopyron elongatum grass and Tetraploid Elytrigia bi-material have again onesize PCR product simultaneously, and the clip size of PCR product is greater than 500bp, good reproducibility, band stable and the PCR product is clear.
From 36 RAPD primers (giving birth to worker's biotechnology company limited by Shanghai synthesizes), filter out special primer LW10 (shown in the SEQ.ID.NO.1).
1.3 get each 5 μ l of amplified production, all electrophoresis detection in containing 1.2% agarose of 1mg/ml EB is observed and the photographic recording result under ultraviolet imagery system (Gel Doc-2000, Bio-Rad, USA).
1.4 carrier construction also transforms
The agarose electrophoresis product is reclaimed and purifying, then (bright restriction enzyme site is EcoR I the fragment that increases to be connected to the pMD18-T carrier, HindIII), then the carrier transfection JM109 competent cell after will connecting, be inoculated on the LB solid medium flat board that contains penbritin (50 μ g/ml), 0.1MIPTG the X-gal 40 μ l of 4 μ l and 20mg/ml induce 37 ℃ of incubator incubated overnight;
The a small amount of hickie bacterium colony of picking carries out pcr amplification, simultaneously it is rule enlarged culturing, 37 ℃ of overnight incubation at the LB solid medium flat board that contains penbritin (50 μ g/ml);
1.5 choose the bacterium after the enlarged culturing, extract DNA, then carry out pcr amplification, the primer is that universal primer is RV-M, M13-47 (Kang, 2005), its amplification system is as follows:
94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 60s, 35 circulations; 72 ℃ are extended 10min.
1.6 the fragment of amplification is checked order, its nucleotide sequence is shown in SEQ.ID.No.2, in NCBI the fragment (being called LW10) of amplification being carried out Blast searches for, utilize the softwares such as Primer Premier 5.0 and DNAMAN 5.0 to carry out sequence alignment and analysis, the result shows, 1305 bases in it and the chromosome of wheat 3B specific B AC storehouse have 84% homology, and its analytical results also can be called this fragment long fringe couchgrass E group chromosome specific fragment as shown in Figure 2.
This fragment is the specific fragment that derives from long fringe couchgrass E group chromosome, it can screen the offspring of containing long fringe couchgrass E group chromosome, this fragment requires to have low homology under the wheat genetic background, it is advantageous that, it can screen the offspring of containing long fringe couchgrass E group chromosome under the wheat genetic background more efficiently and accurately, easier, the easy row of the process that the offspring is screened, reliable.
2, RAPD is converted into the SCAR mark
Requirement according to design SCAR primer: (1) primer length scope 18-26bp, normally select 20-22bp; (2) annealing region is 50～60 ℃, is preferably in 52～58 ℃; (3) GC content range 40-55%; (4) product clip size 400～800bp.
Utilize the softwares such as Primer Premier 5.0 and DNAMAN 5.0 to carry out the special SCAR design of primers of RAPD fragment, design result is as follows:
2C-F
839: GACGCAACAATAACCCTC (shown in the SEQ.ID No.3)
2C-R
839: GTTTGCTTGACTGGCTTG (shown in the SEQ.ID No.4)
The reaction system and the response procedures that carry out pcr amplification with this primer pair are respectively:
PCR reaction system: dna profiling 30ng, 10 * PCR buffer, 2.0 μ l, dNTP Mix (2.5mM) 1.5 μ l, upstream primer (10 μ M) 1 μ l, downstream primer (10 μ M) 1 μ l, r Taq polymerase (5U/ μ l) 0.2 μ l, the sterilization ultrapure water complements to 20 μ l;
The amplification program of described pcr amplification is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 60s, 30～35 circulations; 72 ℃ are extended 10min.
Described pcr amplification product clip size is 839bp, so with its called after SCAR
839, concrete nucleotide sequence is shown in SEQ.ID No.5.
3, SCAR
839The molecule marker checking
3.1 the special SCAR of long fringe couchgrass
839Molecule marker
Extract the DNA of common wheat " China spring ", " China spring "-gametocidal chromosome 2C disomic addition line, Diploid Thinopyron elongatum grass, Tetraploid Elytrigia material, take its genome as template, adopt 2C-F
839And 2C-R
839As primer pair amplification, again its amplified production is carried out gel electrophoresis, carried out the checking of SCAR mark, concrete detected result as shown in Figure 3:
In Diploid Thinopyron elongatum grass (swimming lane 2), Tetraploid Elytrigia (swimming lane 3), amplified the fragment of 839bp, and in common wheat " China spring " (swimming lane 1), " China spring "-gametocidal chromosome 2C disomic addition line (swimming lane 4) this purpose band not, this mark of preliminary proof can be used for the detection of long fringe couchgrass E group chromosome.
3.2 the special SCAR of long fringe couchgrass
839The molecule marker location
Utilization " China spring "-long fringe couchgrass 1E～7E one cover disomic addition line is to SCAR
839Molecule marker positions, and respectively take the genome of 1E～7E disomic addition line as template, adopts 2C-F
839And 2C-R
839Increase as primer pair, again its amplified production is carried out gel electrophoresis, carried out SCAR mark location, concrete detected result is as shown in Figure 4: wherein swimming lane 9 positive contrasts, can see that this mark is positioned on two karyomit(e)s with ancestral group of " China spring "-long fringe couchgrass 2E (swimming lane 2) and 3E (swimming lane 3).
3.3 the special SCAR of long fringe couchgrass
839Molecule marker is used
Further utilize SCAR
839Molecule marker is to the hybrid F of " China spring "-gametocidal chromosome 2C disomic addition line with " China spring "-long fringe couchgrass 3E disomic addition line
1Generation, F
2The generation analyze, electrophoresis result as shown in Figure 5:
" China spring " as negative control do not amplify the purpose fragment, and positive control Diploid Thinopyron elongatum grass amplifies the purpose fragment, at whole F
1For (swimming lane 1～9) and part F
2For having amplified the purpose fragment, this SCAR in the material (swimming lane 10～19)
839The genomic characteristic of the detected result of mark and material matches, and shows the specificity of detection; Further proved this SCAR
839Mark can be used for detecting the long fringe couchgrass E group chromosome under the Wheat Background.
Therefore this SCAR mark just can be applicable to long fringe couchgrass 2E or the 3E Chromosome Identification under the wheat genetic background or follows the tracks of and detect:
Carry out pcr amplification with the upstream and downstream primer pair testing gene group shown in SEQ.ID.NO.3, the SEQ.ID.NO.4, then according to the electrophoresis result of amplified production, judge whether containing long fringe couchgrass E group chromosome: if contain the target fragment of 839bp in the middle of the electrophoresis result of amplified production, then contain long fringe couchgrass E group chromosome in the testing gene group, and be 2E or 3E karyomit(e); If there is not the target fragment of 839bp in the middle of the electrophoresis result of amplified production, then not long fringe couchgrass 2E or 3E karyomit(e) in the testing gene group.
This SCAR mark is also had laid a good foundation for the long fringe couchgrass 2E under the Rapid identification wheat genetic background or 3E chromatin or karyomit(e), and the while also provides very convenient efficiently approach for molecular mark: screening criteria namely contains long fringe couchgrass 2E or 3E chromatin or chromosomal offspring under common wheat " China spring " background.
Claims (7)
1. a marker gene of identifying long fringe couchgrass E group chromosome is characterized in that, its nucleotide sequence is shown in SEQ.ID.NO.2 or SEQ.ID.NO.5.
2. the primer pair of the mark shown in the SEQ.ID.NO.5 that increases is characterized in that, the nucleotide sequence of its upstream and downstream primer is respectively shown in SEQ.ID.NO.3, SEQ.ID.NO.4.
3.SEQ.ID.NO.5 shown sequence is as the long fringe couchgrass 2E of SCAR tag application under the wheat genetic background or the application of the chromosomal evaluation of 3E or tracking detection.
4. application as claimed in claim 3 is characterized in that, is applied to the molecular mark based on long fringe couchgrass 2E or 3E karyomit(e) system cultivation wheat breed.
5. application as claimed in claim 3, it is characterized in that, it is to carry out pcr amplification with the upstream and downstream primer pair testing gene group shown in SEQ.ID.NO.3, the SEQ.ID.NO.4 that described evaluation or tracking detect, then according to the electrophoresis result of amplified production, to whether containing long fringe couchgrass 2E or 3E karyomit(e) is judged.
6. application as claimed in claim 5 is characterized in that, the program of described pcr amplification is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 60s, 30～35 circulations; 72 ℃ are extended 10min.
7. application as claimed in claim 5 is characterized in that, if contain the target fragment of 839bp in the middle of the electrophoresis result of amplified production, then contains long fringe couchgrass 2E or 3E karyomit(e) in the testing gene group; If there is not the target fragment of 839bp in the middle of the electrophoresis result of amplified production, then not long fringe couchgrass 2E or 3E karyomit(e) in the testing gene group.
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| CN103243164A (en) * | 2013-05-06 | 2013-08-14 | 西北农林科技大学 | Method for identifying psathyrostachys huashanica allogenic material in wheat genome |
| CN110029188B (en) * | 2019-04-29 | 2020-04-14 | 四川农业大学 | Tetraploid elytrigia elongata 1E chromosome molecular marker and application thereof |
| CN110643731B (en) * | 2019-10-30 | 2023-06-09 | 山西省农业科学院作物科学研究所 | Rapid detection of Elytrigia intermedium 6J S Specific molecular marker of chromosome and application |
| CN113355449B (en) * | 2021-07-12 | 2022-07-01 | 中国科学院成都生物研究所 | Elytrigia elongata 3E chromosome specific codominant KASP molecular marker and application thereof |
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