CN102443639B - TRAP (Telomeric Repeal Amplification Protocol)-SCAR (Sequence Characterized Amplified Region) 424 marker for identifying E genome of agropyron elongatum and application of marker - Google Patents
TRAP (Telomeric Repeal Amplification Protocol)-SCAR (Sequence Characterized Amplified Region) 424 marker for identifying E genome of agropyron elongatum and application of marker Download PDFInfo
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Abstract
The invention discloses a TRAP (Telomeric Repeal Amplification Protocol)-SCAR (Sequence Characterized Amplified Region) 424 marker for identifying E genome of agropyron elongatum and application of the marker. The nucleotide sequence of the marker is shown by SEQ.ID.NO.3 or SEQ.ID.NO.6. A specific TRAP segment is obtained by using a TRAP-SCAR method through screening and is converted into a stable SCAR marker; and the amplification of the stable SCAR marker is detected. According to the marker for the E genome of the agropyron elongatum which is provided by the invention, through the detection of the SCAR marker, favorable foundation for identifying E chromatin or genome of the agropyron elongatum under the genetic background of wheat is laid; and meanwhile, a very convenient path for molecular marker assistant breeding is provided.
Description
Technical field
The invention belongs to the technical field that long fringe couchgrass E group chromosome is identified, relate to a kind of TRAP-SCAR that identifies long fringe couchgrass E group chromosome
424mark and application thereof.
Background technology
Wheat is one of important in the world food crop.In China, it is the second largest farm crop that are only second to paddy rice, and Wheat Production has very important strategic importance to the national economic development.Due to the Wheat cultivar simplification, make its hereditary basis day by day narrow, anti-source scarcity, 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 to wheat always.Genus Agropyron is that a sibling species of wheat belongs to, it has many good characters that common wheat lacks, as strong 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 stripe rust of wheat, leaf rust, black rust, Powdery Mildew, yellow dwart, stripe mosaic disease and bunt high resistance, to immunity, is one of important parent of wheat genetic breeding.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 forms, 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 have successfully been shifted abroad from long fringe couchgrass.Domesticly little lay down No. 4, No. 5, No. 6, the little wheat breeds such as 107 of laying down have been bred as from wheat and long fringe couchgrass hybrid generation.
By hybridization and the mode of gradually oozing, be by a kind of effective means of the good character of exogenous chromosome and excellent gene importing wheat, 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 agronomy and biochemical analysis by valuable genetic mapping on long fringe couchgrass karyomit(e), but the detection means of these experiments is unsettled, and be not suitable for carrying out the detection of important agronomy character.Some genetics means are arranged, 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 by these laboratory facilities and still have certain limitation.So an urgent demand exploitation is a kind of can identify fast, accurately, on a large scale long fringe couchgrass external source chromatin or chromosomal molecular marking technique under the wheat genetic background.
Target position zone amplification polymorphism (TRAP) is a kind of molecule marker of novel PCR-based, by USDA north crop science laboratory Hu and Vick, in 2003, proposed, a large amount of sequence informations of many biologies that it produces by the large scale sequencing technology, utilize information biology instrument and expressed sequence tag (expressed sequence tag, EST) database information design primer, target candidate gene order district is increased and produces polymorphic molecular marker, this labeling technique has been widely used in vegeto-animal research, the main characterization and evaluation in germ plasm resource, the structure of genetic map, draw the genome transcripting spectrum, important proterties mark, comparative genomics, the aspect such as sibship and genetic diversity has obtained progress.At present, this mark application of having succeeded in the plants such as paddy rice, barley, wheat, Kidney bean, Sunflower Receptacle, beet.
Summary of the invention
The problem that the present invention solves is to provide a kind of TRAP-SCAR that identifies long fringe couchgrass E group chromosome
424mark 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:
A kind of marker gene of identifying long fringe couchgrass E group chromosome, its nucleotide sequence is as shown in SEQ.ID.NO.3 or SEQ.ID.NO.6.
A kind of primer pair of the mark shown in SEQ.ID.NO.3 that increases, the nucleotide sequence of its upstream and downstream primer is respectively as shown in SEQ.ID.NO.1, SEQ.ID.NO.2.
A kind of primer pair of the mark shown in SEQ.ID.NO.6 that increases, the nucleotide sequence of its upstream and downstream primer is respectively as shown in SEQ.ID.NO.4, SEQ.ID.NO.5.
The evaluation of the long fringe couchgrass E group chromosome under the wheat genetic background as the SCAR tag application of the sequence shown in SEQ.ID.NO.6 or tracking detect.
The described molecular mark that is applied to cultivate based on long fringe couchgrass E group chromosome system 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.4, SEQ.ID.NO.5, then according to the electrophoresis result of amplified production, to whether containing long fringe couchgrass E group chromosome, 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 424bp in the middle of the electrophoresis result of amplified production, in the testing gene group, contain long fringe couchgrass E group chromosome; If there is no the target fragment of 424bp in the middle of the electrophoresis result of amplified production, not long fringe couchgrass E group chromosome in the testing gene group.
Compared with prior art, the present invention has following useful technique effect:
1, the present invention adopts a plurality of combination of primers to utilize the analysis of TRAP molecular marking technique to the polymorphism of " China spring " wheat, Diploid Thinopyron elongatum grass, the three kinds of long fringe couchgrass of material E group chromosomes of Tetraploid Elytrigia, filter out the TRAP 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 detected, 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 and detect.Compare with existing authentication method, utilize this SCAR mark to be identified that accuracy will obviously promote undoubtedly, and be very convenient, fast.
2, the SCAR mark that the present invention obtains is called SCAR
424utilize 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, amplified the fragment of 424bp in Diploid Thinopyron elongatum grass, Tetraploid Elytrigia, and in common wheat " China spring ", " China spring "-gametocidal chromosome 2C disomic addition line this product not, this mark of preliminary proof can be for the detection of long fringe couchgrass E group chromosome.
Further use this hybrid F to SCAR primer pair " China spring "-gametocidal chromosome 2C disomic addition line and " China spring "-long fringe couchgrass 7E disomic addition line
1generation, F
2in generation, analyzed, and result is at whole F
1generation and part F
2for having amplified the purpose fragment in material, further proved this SCAR
424mark can be used for detecting the long fringe couchgrass E group chromosome under Wheat Background, for the quick tracking of long fringe couchgrass E group chromosome in Wheat Background detects, provides new way.
3, the detection of SCAR mark used, the PCR reaction system of use, do not need special PCR reaction kit and special reaction reagent, with other common PCR reaction, requires the same; Only need to after pcr amplification, pass through electrophoresis detection, just can have or not the identical purpose band of size to be judged 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 Rapid identification wheat genetic background, also for molecular mark, provide very convenient approach efficiently simultaneously.
The accompanying drawing explanation
Fig. 1 is the amplification of TRAP primer 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 SCAR
424be marked at four kinds of amplifications in differing materials, swimming lane 1 is " China spring ", swimming lane 2 is " China spring "-gametocidal chromosome 2C disomic addition line, and swimming lane 3 is the Diploid Thinopyron elongatum grass, and swimming lane 4 is Tetraploid Elytrigia (Marker:DL2000);
Fig. 3 is SCAR
424the positioning result of molecule marker in a set of disomic addition line of " China spring "-long fringe couchgrass 1E～7E, swimming lane 1 is " China spring ", swimming lane 2 is " China spring "-gametocidal chromosome 2C disomic addition line, swimming lane 3 is the Diploid Thinopyron elongatum grass, swimming lane 4 is " China spring "-long fringe couchgrass 1E disomic addition line, swimming lane 5 is " China spring "-long fringe couchgrass 2E disomic addition line, swimming lane 6 is " China spring "-long fringe couchgrass 3E disomic addition line, swimming lane 7 is " China spring "-long fringe couchgrass 4E disomic addition line, swimming lane 8 is " China spring "-long fringe couchgrass 5E disomic addition line, swimming lane 9 is " China spring "-long fringe couchgrass 6E disomic addition line, swimming lane 10 is " China spring "-long fringe couchgrass 7E disomic addition line (Marker:DL2000),
Fig. 4 is SCAR
424be marked at " China spring "-long fringe couchgrass 7E disomic addition line and " China spring "-gametocidal chromosome 2C disomic addition line hybrid generation F
1, F
2in amplification, swimming lane 1 is common wheat-" China spring ", swimming lane 2 is " China spring "-gametocidal chromosome 2C disomic addition line, swimming lane 3 is the Diploid Thinopyron elongatum grass, and swimming lane 4～13 is that " China spring "-long fringe couchgrass 7E disomic addition line hybridizes with " China spring "-gametocidal chromosome 2C disomic addition line the F obtained
1for material, swimming lane 14～24 is F
1the F obtained for selfing
2for material (Marker:DL2000).
Embodiment
The invention provides a kind of TRAP-SCAR that identifies long fringe couchgrass E group chromosome
424mark and application thereof, obtain specific TRAP fragment by the screening of TRAP-SCAR method, and be converted to stable SCAR mark, and its amplification is detected.Preparation, amplification and evaluation below in conjunction with specific embodiment to mark are described in further detail, and 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 by 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 by 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, TRAP molecule marking method screening specificity T RAP fragment
Utilize the TRAP molecule marking method, carry out the screening of polymorphism in common wheat " China spring ", Diploid Thinopyron elongatum grass, Tetraploid Elytrigia, obtain specific fragment.
1.1 adopt the CTAB method to extract respectively genomic dna from fresh differing materials young leaflet tablet, and detect DNA concentration and quality;
1.2TRAP fragment amplification
PCR reaction system: DNA profiling 50ng, 10 * PCR buffer, 2.0 μ l, dNTP Mix (2.5mM) 1.5 μ l, arbitrary primer (0.3pmol) 1 μ l, immobilized primer (10pmol) 1 μ l, r Taqpolymerase (5U/ μ l) 0.2 μ l, the sterilizing ultrapure water complements to 20 μ l;
The PCR response procedures:
This response procedures is before 5 of starting most circulate in, then 35 circulate in after.
In the process of screening, utilize common wheat " China spring ", Diploid Thinopyron elongatum grass, three kinds of materials of Tetraploid Elytrigia to be screened these 168 combination of primers, screening according to being 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, reproducible, band stable and the PCR product is clear.
14 forward primers derive from Zhang Na (2009), Li and Klindworth (2006), 12 reverse primers derive from Zhang Na (2009), Li and Quiros (2001), primer is synthetic by Shanghai Sheng Gong company, and forward primer and reverse primer have 168 combination of primers.Therefrom filter out special primer to W09ARBI3 (shown in SEQ.ID.NO.1 and SEQ.ID.NO.2)
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 of amplification to be connected to the pMD18-T carrier, HindIII) on, then the carrier transfection JM109 competent cell after connecting, be inoculated on the LB solid medium flat board that contains penbritin (50 μ g/ml), 0.1M the X-gal 40 μ l of IPTG 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, it rule on the LB solid medium flat board that contains penbritin (50 μ g/ml) simultaneously, and enlarged culturing, 37 ℃ of overnight incubation;
1.5 choose the bacterium after 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 as shown in SEQ.ID No.3, in NCBI, the fragment (being called W09 ARBI3) of amplification is carried out to the Blast search, utilize the softwares such as Primer Premier5.0 and DNAMAN 5.0 to carry out sequence alignment and analysis, result shows, do not there is with it the sequence of homology, this fragment can be called to long fringe couchgrass E group chromosome specific fragment yet.
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 can make the offspring screen, reliable.
2, TRAP is converted into to 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 TRAP fragment, design result is as follows:
2C-F
424: CAAATGGTGGTGGAGGAT (shown in SEQ.ID No.4)
2C-R
424: TATGAGGCGAGGCAACAG (shown in SEQ.ID No.5)
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 sterilizing 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 424bp, so by its called after SCAR
424, concrete nucleotide sequence is as shown in SEQ.ID No.6.
3, SCAR
424the molecule marker checking
3.1 the special SCAR of long fringe couchgrass
424molecule 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
424and 2C-R
424as primer pair amplification, then its amplified production is carried out to gel electrophoresis, has carried out the checking of SCAR mark, concrete detected result as shown in Figure 2:
Amplified the fragment of 424bp in Diploid Thinopyron elongatum grass (swimming lane 3), Tetraploid Elytrigia (swimming lane 4), and in common wheat " China spring " (swimming lane 1), " China spring "-gametocidal chromosome 2C disomic addition line (swimming lane 2) this purpose band not, this mark of preliminary proof can be for the detection of long fringe couchgrass E group chromosome.
3.2 the special SCAR of long fringe couchgrass
424the molecule marker location
Utilize a set of disomic addition line of " China spring "-long fringe couchgrass 1E～7E to SCAR
424molecule marker positions, and the genome of 1E～7E disomic addition line of take respectively is template, adopts 2C-F
424and 2C-R
424as primer pair, increase, again its amplified production is carried out to gel electrophoresis, carried out SCAR mark location, concrete detected result is as shown in Figure 3: the wherein positive contrast of swimming lane 3, can see that this mark is positioned 7 of " China spring "-long fringe couchgrass 1E～7E with on all karyomit(e)s of ancestral group (shown in swimming lane 4～10).
3.3 the special SCAR of long fringe couchgrass
424the molecule marker application
Further utilize SCAR
424molecule marker is the hybrid F with " China spring "-long fringe couchgrass 7E disomic addition line to " China spring "-gametocidal chromosome 2C disomic addition line
1generation, F
2the generation analyze, electrophoresis result as shown in Figure 4:
" 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 4～13) and part F
2for having amplified the purpose fragment in material, this SCAR
424the genomic characteristic of the detected result of mark and material matches, and shows the specificity of detection; Further proved this SCAR
424mark can be used for detecting the long fringe couchgrass E group chromosome under Wheat Background.
Therefore the long fringe couchgrass E group chromosome that this SCAR mark just can be applicable under the wheat genetic background is identified or is followed the tracks of and detect:
Carry out pcr amplification with the upstream and downstream primer pair testing gene group shown in SEQ.ID.NO.4, SEQ.ID.NO.5, then according to the electrophoresis result of amplified production, to whether containing long fringe couchgrass E group chromosome, judged: if the target fragment that contains 424bp in the middle of the electrophoresis result of amplified production contains long fringe couchgrass E group chromosome in the testing gene group; If there is no the target fragment of 424bp in the middle of the electrophoresis result of amplified production, not long fringe couchgrass E group chromosome in the testing gene group.
This SCAR mark is also had laid a good foundation for long fringe couchgrass E group chromatin or karyomit(e) under Rapid identification wheat genetic background, and also for molecular mark, provide very convenient approach efficiently: screening criteria contains long fringe couchgrass E group chromatin or chromosomal offspring under common wheat " China spring " background simultaneously.
Claims (7)
1. a marker gene of identifying long fringe couchgrass E group chromosome, is characterized in that, its nucleotide sequence is as shown in SEQ.ID.NO.3 or SEQ.ID.NO.6.
2. the primer pair of the mark shown in SEQ.ID.NO.6 that increases, is characterized in that, the nucleotide sequence of its upstream and downstream primer is respectively as shown in SEQ.ID.NO.4, SEQ.ID.NO.5.
3.SEQ.ID.NO.6 the application that the evaluation of shown sequence long fringe couchgrass E group chromosome under the wheat genetic background as the SCAR tag application or tracking detect.
4. application as claimed in claim 3, is characterized in that, is applied to cultivate based on long fringe couchgrass E group chromosome system the molecular mark of 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.4, SEQ.ID.NO.5 that described evaluation or tracking detect, then according to the electrophoresis result of amplified production, to whether containing long fringe couchgrass E group chromosome, 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 the target fragment that contains 424bp in the middle of the electrophoresis result of amplified production contains long fringe couchgrass E group chromosome in the testing gene group; If there is no the target fragment of 424bp in the middle of the electrophoresis result of amplified production, not long fringe couchgrass E group chromosome in the testing gene group.
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