CN101165193A - EST-STS marking primer 1 with differential rye1RS chromosome, screening method and use - Google Patents
EST-STS marking primer 1 with differential rye1RS chromosome, screening method and use Download PDFInfo
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Abstract
The present invention provides one kind of specific EST-STS marking primer-1 of rye 1RS chromosome. The marking primer-1 is designed with the EST sequence, BE637935, of the first partial wheat homologous group as the template, and has the sequences including STSWE126F of 5'-TCAAGCACGCATTTCAACTC-3' and STSWE126R of 5'-ACAGATGTCCAAAGCCCAAC-3'. The primer STSWE126 has one amplified 850 bp size specific segment located in rye 1RS chromosome. The marking primer-1 of the present invention expands the use range of wheat EST, provides one new tool for deep research of distant hybrid material for common wheat. The marking primer-1 may be applied in identifying rye 1RS chromosome simply and fast by means of common PCR technology.
Description
Technical field
The present invention relates to special EST-STS labeled primer 1, screening method and the purposes of a kind of rye 1RS karyomit(e), belong to the gene molecule marker research field.
Background technology
Wheat is one of important crops in the world, usually is subjected to the threat of various diseases aborning, causes the decline of output or quality.The nearly edge species rye of wheat has disease-resistant, high yield, anti-many good characters such as lean.Studies show that has numerous resistant genes such as mildew-resistance, stripe rust resisting, leaf rust resistance, anti-black rust, anti-green bugs on the rye 1RS karyomit(e), improve output and adaptive gene in addition.By distant hybirdization and karyomit(e) operative technique, these desirable genes on the rye 1RS karyomit(e) can be directed into common wheat.
Rye 1RS karyomit(e) or chromosome segment need be identified it after importing common wheat, just can effectively utilize the excellent gene of external source on the 1RS.Be based upon the molecule marker on the dna sequence dna basis,, accurately, fast, be not subjected to advantages such as plant-growth restriction in period with it and extensively used particularly based on the molecule marker of PCR.Ko Jong-Min etc. has filtered out RAPD (the Random amplified polymorphicDNA of two rye genome specifics, the polymorphic dna of random amplification) mark, and two clones pSc10C and pSc20H have been obtained, the in situ hybridization result shows that they are distributed in (reference: Jong-Min Ko on all chromosomess of rye, Geum-Sook Do, Duck-Yong Suh, et al.2002.Identification and chromosomal orgnization of two rye genome-specifiRAPD products useful as introgression markers in wheat.Genome, 45:157-164).Yet RAPD mark less stable is difficult to effectively utilize in breeding.M.Cristina Katto etc. have designed according to the pSc20H clone can identify the segmental mark based on PCR of chromosomes of rye (reference: M.Cristina Katto, Takashi R.Endo, Shuhei Nasuda.2004.A PCR-based marker for targeting for small ryesegments in wheat background.Genes Genet.Syst., 79.p.245-250).Liu Cheng etc. screen the special RAPD mark of rye, and (reference: Liu becomes to be translated into the SCAR mark, Li Guangrong, poplar foot monarch, Feng Juan, Zhou Jianping appoints the separation of just grand .2006. rye genome specific dna fragmentation and the foundation of SCAR mark. northwest Botany Gazette, 26 (12): 2434-2438).Yet these marks all have amplification to the full genome of rye, are not that 1R or 1RS karyomit(e) are special, thereby can't be with 1R karyomit(e) and the 2R of rye
～The 7R chromosomal region separately.Koebner etc. are according to rye and wheat rRNA intergenic region sequence difference, synthesized rye 1R karyomit(e) special primer NOR-R1, can identify 1RS karyomit(e), but to the recombined material that do not contain nucleolus organizer region on the 1R karyomit(e) with primer NOR-R1 with fubaritic (reference: Koebner R.M.D.1995.Generation of PCR-based markers for thedetection of rye chromatin in wheat background.Theor Appl Genet, 90:740-745).
EST (expressed sequence tag, expressed sequence tag) is meant random choose clone from the cDNA library of different tissue sources, and its 3 ' end or 5 ' end carried out the short cDNA sequence (reference: Adams M D that single-wheel order-checking is obtained, Kelley J M, Gocayne JD, Dubnick M, Polymeropoulos M is DNAsequencing:expressed sequence tags and human genome project.Science H.1991.Complementary, 252:1651-1656).Because the EST mark is directed to cDNA, its conservative property is higher, particularly between affinity species, has relative conservative property, so according to STS mark (the Site tagged sequence that EST developed, the site sequence label, promptly directly at est sequence two ends designs primer) between species, have a good versatility (reference: LaRota M, Sorrells M is DNA sequence analysis of mappedwheat ESTs reveals the complexity of genome relationships betweenwheat and rice.Funct Integr Genomics E.2004.Comparative, 4:34-46).Comparative genomics studies show that between common wheat and the nearly edge species thereof and has collinearity, therefore can utilize the est sequence of having developed on the wheat to study the affinity species of wheat.
Summary of the invention
The present invention utilizes Secale (Secale) and Triticum (Triticum) to belong to wheat subtribe (Triticinae), they have higher collinearity relation in theory, the est sequence that is positioned on the homology group of wheat first part also is present in the chromosomal corresponding site of rye 1R probably, therefore design is based on the mark of PCR, change into the STS primer, the special molecule marker of screening rye 1RS karyomit(e) is with the rye 1RS karyomit(e) in rapid detection and the tracking importing general wheat background.
Technical scheme of the present invention realizes by following means: the special EST-STS labeled primer 1 of this rye 1RS karyomit(e), it is characterized in that: this labeled primer is that the est sequence-BE637935 according to the homology group of wheat first part is that stencil design obtains, and its sequence is:
STS
WE126：F：5’-TCAAGCACGCATTTCAACTC-3’，
R：5’-ACAGATGTCCAAAGCCCAAC-3’。
The special EST-STS labeled primer 1 of described rye 1RS karyomit(e) is characterized in that: primer STS
WE126The size that amplifies is that the specific fragment of 850bp is positioned on the rye 1RS karyomit(e).
The screening method of the EST-STS labeled primer 1 that described rye 1RS karyomit(e) is special, its feature comprises the steps:
A, primer design and synthetic: according to the homology group's of wheat first part est sequence, choosing 35 base numbers downloads greater than the est sequence of 300bp, the 35 pairs of primers that utilized PrimerPremier 5.0 software designs, condition is annealing temperature 50-60 ℃, the length 18-22bp of primer, GC% content is 40-60%, expection amplified production 300-600bp; The synthetic primer is dissolved to 20 μ mol L with ultrapure water
-1Preserve in-20 ℃ of refrigerators as mother liquor, it is stand-by as working fluid to use preceding absorption partial mother liquid to be diluted to 2 μ mol/L with 10mM Tris-HCl and 1mM EDTA;
The screening of b, chromosomes of rye specific mark primer: utilize 35 pairs of EST-STS primers of step a design synthetic, respectively to the common wheat China spring; The genomic dna of the nearly edge species of wheat kingdom rye, cluster hair wheat, barley and roegneria kamoji carries out the polymorphism amplification to be analyzed, as a result primer STS
WE126, sequence is: STS
WE126F:5 '-TCAAGCACGCATTTCAACTC-3 ' and ST
SWE126R:5 '-ACAGATGTCCAAAGCCCAAC-3 ' _ in the kingdom rye, amplify the special band that a size is 850bp, thus filter out the special EST-STS labeled primer of chromosomes of rye group;
The screening of c, rye 1R karyomit(e) specific mark primer: utilize the polymorphism EST-STS primer that screens among the step b, respectively to the analysis of increasing of the disomic addition line of common wheat China spring and a whole set of China spring-kingdom rye, the result amplifies the special band that a size is 850bp respectively in kingdom rye and the China spring-rye 1R of kingdom disome alien addition line, determine rye 1R karyomit(e) specific mark primer;
The screening of d, rye 1RS karyomit(e) specific mark primer: utilize the polymorphism EST-STS primer that screens among the step b, respectively lay down for a short time No. 6, kingdom rye, common wheat of common wheat are engraved virtuous 169 and wheat/rye 1BL/1RS translocation line Luo Fulin 10, Luo Fulin 13 analysis of increasing, Luo Fulin 10, Luo Fulin 13 and kingdom rye can amplify the special band that a size is 850bp as a result, determine rye 1RS karyomit(e) specific mark primer STS
WE126
The screening method of the EST-STS labeled primer that described rye 1RS karyomit(e) is special, the described amplification analysis of step b comprises: add the template DNA of about 10～20ng successively in volume is the Eppendof pipe of 0.2mL, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L-1dNTP, left and right sides primer final concentration respectively are 0.2 μ mol L
-1, 0.5U Taq archaeal dna polymerase is used sterile distilled water postreaction system to 10 μ L at last; To there be the Eppendof pipe of above-mentioned reaction solution to be placed on then and carry out amplified reaction on the PE2700 gene-amplificative instrament; The amplified reaction program is: 94 ℃ of pre-sex change of elder generation 3 minutes; 94 ℃ of sex change are 30 seconds again, 55 ℃ of annealing 30 seconds, and 72 ℃ were extended 1 minute and 20 seconds, and circulated 32 times; 72 ℃ were extended 10 fens; Last 10 ℃ of preservations;
Amplified reaction finishes the back pcr amplification product is carried out electrophoresis detection, as a result primer STS
WE126The band that to amplify a size in the kingdom rye be 850bp does not amplify this band and have the genomic China spring of rye, barley, cluster hair wheat and roegneria kamoji, determines ST
SWE126It is exactly the special mark of chromosomes of rye group.
The screening method of the EST-STS labeled primer 1 that described rye 1RS karyomit(e) is special, described pcr amplification product carries out the electrophoresis detection step: add indicator 2 μ L in pcr amplification product, mixing, getting wherein, 3 μ L detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, behind the electrophoresis 1.5h, dye with argentation, the glue after will dyeing is at last observed photograph on the gel imaging instrument.
The purposes of the EST-STS labeled primer 1 that described rye 1RS karyomit(e) is special is characterized in that: the applying marking primer amplification is analyzed 1RS transposition material, can amplify the material of a special band of 850bp, can determine to contain rye 1RS karyomit(e).
The present invention compared with the prior art advantage is:
1, the special EST-STS labeled primer of rye 1RS karyomit(e) of the present invention has not only been widened the use range of wheat EST, provides new instrument for further investigation common wheat remote hybrid material simultaneously.
2, the special EST-STS labeled primer of rye 1RS karyomit(e) of the present invention utilizes the regular-PCR technology just can carry out, and does not need other red tapes, can identify rye 1RS karyomit(e) simply, apace.
3, the binding ability of special EST-STS labeled primer of rye 1RS karyomit(e) of the present invention and template is stronger, though this studies employed annealing temperature is 55 ℃, but annealing temperature all can amplify target fragment preferably at 51-58 ℃, and requirement for experiment condition is comparatively loose.
4, the special EST-STS labeled primer sequence of rye 1RS karyomit(e) of the present invention is positioned at wheat 1B karyomit(e) near the zone, kinetochore, principle according to comparative genomics, this mark also should be positioned at 1R karyomit(e) near the zone, kinetochore, therefore can identify the material that relates to the different breakpoints of 1RS in conjunction with the chromosomal specific mark of other 1RS.
Description of drawings
The sequence of Fig. 1: BE637935 and STS of the present invention
WE126Forward primer and the position of reverse complementary sequence on est sequence of reverse primer.
Fig. 2: design 35 pairs of EST-STS primers of synthetic respectively to the common wheat China spring with the present invention; The genomic dna of the nearly edge species of wheat kingdom rye, cluster hair wheat, barley and roegneria kamoji carries out the polymorphism amplification to be analyzed, and the result has only primer STS
WE126In the kingdom rye, amplify size and be the band of 850bp, and China spring, barley, cluster hair wheat and roegneria kamoji do not amplify this band, and STS is described
WE126It is the special mark of chromosomes of rye group.
Fig. 3: with common wheat China spring, kingdom rye and the China spring-rye 1R of kingdom～7R disome alien addition line the DNA of totally 9 materials be template, with primer STS of the present invention
WE126Carry out pcr amplification, the result shows that have only the chromosomal material of additional 1R can amplify the size the same with the kingdom rye is the special band of 850bp, and contrast China spring and 2R～7R alien addition line do not amplify this special band, illustrate that this band is positioned on the rye 1R karyomit(e).
Fig. 4: with common wheat lay down for a short time No. 6, kingdom rye, common wheat engrave virtuous 169, wheat/rye 1BL/1RS translocation line Luo Fulin 10 and Luo Fulin 13 DNA of totally 5 materials be template, with primer STS of the present invention
WE126Carry out pcr amplification, the result shows that have only Luo Fulin 10 and Luo Fulin 13 can amplify the size the same with the kingdom rye is the special band of 850bp, illustrates that this band is positioned on the rye 1RS karyomit(e).
Fig. 5: with common wheat No. 6 DNA with 11 materials of " Germany white grain " rye filial generation seed selection that lay down for a short time is template, with primer STS of the present invention
WE126Carry out pcr amplification, wherein 4 contain the chromosomal progeny material of 1RS all amplify the size be the special band of 850bp, 7 do not contain the chromosomal material of rye 1RS and do not amplify corresponding special band.Diagram 1～11 is for laying down for a short time No. 6 and " the white grain of Germany " rye filial generation material, and M is molecular weight marker DL2000.
Embodiment
1, the acquisition of EST sequence: according to wheat expressed sequence tag (EST) database of USDA website (http://wheat.pw.usda.gov/GG2/index.shtml) issue, Search and Orientation is in the EST and the sequence thereof of the different physical zone of the homology group of wheat first part, choose 35 conservative propertys more intense and the base number download greater than the est sequence of 300bp.Accompanying drawing 1 is the base sequence of one of them EST-BE637935.
2, primer design and synthetic: the est sequence with downloading in the step 1, utilize PrimerPremier 5.0 softwares that it is carried out primer design, designed 35 pairs of primers altogether.The condition of design of primers is annealing temperature 50-60 ℃, the length 18-22bp of primer, and GC% content (fast Guanine of bird and cytosine(Cyt) Cytosine account for the ratio of base sum) is 40-60%, expection amplified production 300-600bp.The primer of design is synthetic by Shanghai associating gene company limited.
3, the dilution of primer:, be dissolved to 20 μ molL with ultrapure water with synthetic primer in the step 2
-1Preserve in-20 ℃ of refrigerators as mother liquor, draw partial mother liquid before using and be diluted to 2 μ mol L with 1 * TE (10mMTris-HCl and 1mM EDTA PH=8.0)
-1Stand-by as working fluid.
4, the screening of rye specific mark: if certain primer amplifies in rye and other for examination materials discrepant band, this primer is exactly the special primer of the chromosomes of rye group that filters out so.
Utilize 35 pairs of good EST-STS primers of dilution in the step 3, respectively to the common wheat China spring; The genomic dna of the nearly edge species of wheat kingdom rye, cluster hair wheat, barley and roegneria kamoji carries out the polymorphism amplification and analyzes, concrete amplification is as follows: the template DNA that adds about 10～20ng in volume is the Eppendof pipe of 0.2mL successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, left and right sides primer final concentration respectively are 0.2 μ mol L
-1, 0.5U TaqDNA polysaccharase is used sterile distilled water postreaction system to 10 μ L at last.To there be the Eppendof pipe of above-mentioned reaction solution to be placed on then and carry out amplified reaction on the PE2700 gene-amplificative instrament.The amplified reaction program is: 94 ℃ of pre-sex change of elder generation 3 minutes; 94 ℃ of sex change are 30 seconds again, 55 ℃ of annealing 30 seconds, and 72 ℃ were extended 1 minute and 20 seconds, and circulated 32 times; 72 ℃ were extended 10 fens; Last 10 ℃ of preservations.
The pcr amplification product detection method is: add indicator (0.25% tetrabromophenol sulfonphthalein, 0.25% methyl green, 40% sucrose in pcr amplification product, water surplus) 2 μ L, mixing, get wherein 3 μ L with 8% non-denaturing polyacrylamide gel (polyacrylamide: N, N '-methylene diacrylamide=39: 1) electrophoresis detection, electrophoretic voltage are 150～180V, about electrophoresis 1.5h after, dye with argentation, the glue after will dyeing is at last observed photograph on the gel imaging instrument.Primer STS as a result
WE126(sequence is: STS
WE126F:5 '-TCAAGCACGCATTTCAACTC-3 '; STS
WE126R:5 '-ACAGATGTCCAAAGCCCAAC-3 '.) in the kingdom rye, amplify size and be the band of 850bp, and China spring, barley, cluster hair wheat and roegneria kamoji do not amplify this band, as shown in Figure 2, STS are described
WE126It is exactly the special mark of chromosomes of rye group.
5, the screening of rye 1RS karyomit(e) specific mark
The polymorphism EST-STS primer STS that utilizes step 4 to screen
WE126Disome alien addition line to China spring and a whole set of China spring-kingdom rye is (public, reference: Driscoll C J, Sears E R, 1971.Individual additions of the chromosomesof " Imperial " rye to wheat.Agron Abstr, 1971:6) carry out pcr amplification reaction, reaction conditions and amplification program are with step 4.Behind the pcr amplification reaction amplified production is carried out electrophoresis detection, detection method detects the back and determines primer STS with step 4
WE126In kingdom rye and the China spring-rye 1R of kingdom disome alien addition line, amplify the special band that a size is 850bp respectively, and contrast China spring and the China spring-rye 2R of kingdom～7R disome alien addition line all do not amplify this corresponding special band, as shown in Figure 3, illustrate that this is labeled as rye 1R karyomit(e) specific mark.
Utilize primer STS then
WE126Virtuous 169, wheat/rye 1BL/1RS translocation line Luo Fulin 10 and Luo Fulin 13 engraved in lay down for a short time No. 6, kingdom rye, common wheat of amplification common wheat, and reaction conditions and amplification program are with step 4.Behind the pcr amplification reaction to amplified production with carrying out electrophoresis detection, detection method is with step 4, the result shows to have only 1BL/1RS translocation line Luo Fulin 10,1BL/1RS translocation line Luo Fulin 13 and kingdom rye can amplify the special band that a size is 850bp, as shown in Figure 4, illustrate that this amplification site is positioned on the rye 1RS karyomit(e) primer STS
WE126Be the chromosomal specific mark of rye 1RS.Because of it amplifies a special band, so the special EST-STS labeled primer 1 of called after rye 1RS karyomit(e).
The detection in 1RS transposition material of embodiment 2, labeled primer of the present invention is used
Utilize the special labeled primer STS of rye 1RS karyomit(e) that screens among the embodiment 1
WE126, amplification is laid down No. 6 and " the white grain of Germany for a short time
"The part progeny material of rye (S.cereale L.cv germanwhite) hybridization, reaction conditions and amplification program are with step 4, behind the amplified reaction amplified production is detected, detection method is with step 4, and the result is in 11 materials for examination, and 4 materials amplify size and are the special band of 850bp, can determine to contain rye 1RS karyomit(e), other 7 materials do not amplify this corresponding special band, can determine that it does not contain rye 1RS karyomit(e), as shown in Figure 5.This result is consistent with the genomic in situ hybridization qualification result.
The enforcement that the present invention enumerates is intended to further illustrate special EST-STS labeled primer, screening method and the purposes of this rye 1RS karyomit(e).And scope of the present invention is not constituted any restriction.
Claims (6)
1. EST-STS labeled primer 1 that rye 1RS karyomit(e) is special is characterized in that: this labeled primer is that the est sequence-BE637935 according to the homology group of wheat first part is that stencil design obtains, and its sequence is:
ST
SWE126 F：5’-TCAAGCACGCATTTCAACTC-3’，
R：5’-ACAGATGTCCAAAGCCCAAC-3’。
2. according to the special EST-STS labeled primer 1 of the described rye 1RS of claim 1 karyomit(e), it is characterized in that: primer STS
WE126The specific fragment that a size that amplifies is 850bp is positioned on the rye 1RS karyomit(e).
3. according to the screening method of the special EST-STS labeled primer 1 of the described rye 1RS of claim 1 karyomit(e), its feature comprises the steps:
A, primer design and synthetic: according to the homology group's of wheat first part est sequence, choosing 35 base numbers downloads greater than the est sequence of 300bp, the 35 pairs of primers that utilized PrimerPremier 5.0 software designs, condition is annealing temperature 50-60 ℃, the length 18-22bp of primer, GC% content is 40-60%, expection amplified production 300-600bp; The synthetic primer is dissolved to 20 μ mol L with ultrapure water
-1Preserve in-20 ℃ of refrigerators as mother liquor, it is stand-by as working fluid to use preceding absorption partial mother liquid to be diluted to 2 μ mol/L with 10mM Tris-HCl and 1mM EDTA;
The screening of b, chromosomes of rye specific mark primer: utilize 35 pairs of EST-STS primers of step a design synthetic, respectively to the common wheat China spring; The genomic dna of the nearly edge species of wheat kingdom rye, cluster hair wheat, barley and roegneria kamoji carries out the polymorphism amplification to be analyzed, as a result primer STS
WE126(sequence is: STS
WE126F:5 '-TCAAGCACGCATTTCAACTC-3 ' and STS
WE126R:5 '-ACAGATGTCCAAAGCCCAAC-3 ') in the kingdom rye, amplifies size and be the special band of 850bp, thereby filter out the special EST-STS labeled primer of chromosomes of rye group;
The screening of c, rye 1R karyomit(e) specific mark primer: utilize the polymorphism EST-STS primer that screens among the step b, respectively to the analysis of increasing of the disomic addition line of China spring and a whole set of China spring-kingdom rye, the result amplifies size respectively and is the special band of 850bp in kingdom rye and the China spring-rye 1R of kingdom disome alien addition line, determine rye 1R karyomit(e) specific mark primer;
The screening of d, rye 1RS karyomit(e) specific mark primer: utilize the polymorphism EST-STS primer that screens among the step b, respectively lay down for a short time No. 6, kingdom rye, common wheat of common wheat are engraved virtuous 169 and wheat/rye 1BL/1RS translocation line Luo Fulin 10, Luo Fulin 13 analysis of increasing, as a result Luo Fulin 10, Luo Fulin 13 and kingdom rye all can amplify the size be the special band of 850bp, determine rye 1RS karyomit(e) specific mark primer STS
WE126
4. according to the screening method of the special EST-STS labeled primer 1 of the described rye 1RS of claim 1 karyomit(e), it is characterized in that: the described amplification analysis of step b comprises: the template DNA that adds about 10～20ng in volume is the Eppendof pipe of 0.2mL successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, left and right sides primer final concentration respectively are 0.2 μ molL
-1, 0.5U Taq archaeal dna polymerase is used sterile distilled water postreaction system to 10 μ L at last; To there be the Eppendof pipe of above-mentioned reaction solution to be placed on then and carry out amplified reaction on the PE2700 gene-amplificative instrament; The amplified reaction program is: 94 ℃ of pre-sex change of elder generation 3 minutes; 94 ℃ of sex change are 30 seconds again, 55 ℃ of annealing 30 seconds, and 72 ℃ were extended 1 minute and 20 seconds, and circulated 32 times; 72 ℃ were extended 10 fens; Last 10 ℃ of preservations;
Amplified reaction finishes the back pcr amplification product is carried out electrophoresis detection, as a result primer STS
WE126In the kingdom rye, amplify size for the special band of 850bp, do not amplify this band, determine STS and have the genomic China spring of rye, barley, cluster hair wheat and roegneria kamoji
WE126It is exactly the special mark of chromosomes of rye group.
5. according to the screening method of the special EST-STS labeled primer 1 of the described rye 1RS of claim 4 karyomit(e), it is characterized in that: described pcr amplification product carries out the electrophoresis detection step and is: add indicator 2 μ L in pcr amplification product, mixing, getting wherein, 3 μ L detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, behind the electrophoresis 1.5h, dye with argentation, the glue after will dyeing is at last observed photograph on the gel imaging instrument.
6. according to the purposes of the special EST-STS labeled primer 1 of the described rye 1RS of claim 1 karyomit(e), it is characterized in that: the applying marking primer amplification is analyzed 1RS transposition material, the material of the special band of 850bp can be amplified, rye 1RS karyomit(e) can be determined to contain.
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