CN102181548A - Specific molecular markers for 1R-7R chromosomes of rye based on rye EST (expressed sequence tag) sequence and application of specific molecular markers - Google Patents
Specific molecular markers for 1R-7R chromosomes of rye based on rye EST (expressed sequence tag) sequence and application of specific molecular markers Download PDFInfo
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Abstract
The invention discloses 14 specific molecular markers for the 1R-7R chromosomes of rye based on a rye EST (expressed sequence tag) sequence, and the molecular markers are forward primers and reverse primers which are designed according to the EST sequence of rye chromosomes; and the molecular markers are used for identifying the specific markers of the 1R-7R chromosomes of rye. The identification of the 1R-7R chromosomes of rye by applying the molecular markers comprises the basic steps of: A. selecting and preparing the marker primers; B. diluting the marker primers; and C. marking the specificity of the rye chromosomes: C-1, extracting template DNA; C-2, carrying out PCR (polymerase chain reaction) amplification; C-3, detecting an amplified product; and C-4, comparing results. In the invention, a set of the specific molecular markers for the rye chromosomes are designed and developed on the basis of the EST sequence of the rye chromosomes, and the whole 1R-7R rye chromosomes in a genetic background of wheat can be rapidly and accurately identified by using a common PCR technology by means of the molecular markers, and thus the invention provides a simple and effective molecular identification method for the transfer of beneficial genes on the rye chromosomes to wheat cultivation.
Description
Technical field
The present invention relates to molecular biology and genetic breeding field, be specifically related to based on rye expressed sequence tag (expressed sequence tag, EST) molecule marker of sequences Design, and the application of these molecule markers aspect tracking detection chromosomes of rye.
Background technology
The sibling species rye of wheat (
Secale cerealeL.) have many good proterties, as well developed root system, strong, the drought resisting of tillering, barren-resistant, cold-resistant, salt tolerant alkali and acid soil, especially anti-multiple diseases, as wheat powdery mildew (
Blumeria graminisF.sp.
Tritici), stripe rust (
Puccinia graminisF.sp.
Tritici), leaf rust (
P.
TriticinaEriks.)
,Stem rust (
P.
GraminisPers. f. sp.
Tritici), bunt (Tilletia Controversa Kuhn, TCK) and barley yellow dwarf (Barley yellow dwarf virus, BYDV) etc.Therefore, rye is that wheat is carried out genetic improvement, widens the wheat genetic basis, increases the important gene source of wheat cultivation kind heritable variation.
The approach that the excellent gene of rye shifts in wheat mainly is initiative a series of wheats/rye double diploid, addition line, substitution line and translocation line.By means such as distant hybirdization and chromosome engineerings chromosomes of rye is imported in the Wheat Background, afterwards, also need it is identified accurately, could be used effectively.Compare with the biochemical marker method with cytologic technology, molecular labeling method simply, characteristics make it be widely used for detecting, following the tracks of the chromosomes of rye that imports in the Wheat Background fast and accurately.Yet, the specific mark of the chromosomes of rye of present exploitation, on the 1RS karyomit(e) that focuses mostly in rye, as SSR mark Bmac0213(reference: Schneider A and Moln á r-L á ng M. 2008. Polymorphism analysis using 1RS-specific molecular markers in rye cultivars (
Secale cerealeL.) of various origin. Cereal Res Commun, 36:11-19); SSAP mark S10, S20, the S17(reference: Nagy ED and Lelley T. 2003. Genetic and physical mapping of sequence specific amplified polymorphic (SSAP) markers on the 1RS chromosome arm in a wheat background. Theor Appl Genet, 107:1271-1277); EST-STS mark STS
We3And STS
We126(reference: Wang CM, Li LH, Zhang XT, Gao Q, Wang RF, andAn DG. 2009. Development and application of EST-STS markers specific to chromosome 1RS of
Secale cerealeResearch Communications 37:13-21 .Cereal) etc.Zhuang etc. (2008) have reported 1R, 4R, EST-SSR mark (the reference: Zhuang LF that 5R and 7R karyomit(e) are special, Song LX, Feng YG, Qian BL, Xu HB, Pei ZY, Qi ZJ. 2008. Development and chromosome mapping of new wheat EST-SSR markers and application for characterizing rye chromosomes added in wheat. Acta Agron Sin, 34:926-933).Lee etc. (2009) have also reported 6 EST mark (reference: Lee TG that 2RL karyomit(e) is special, Hong MJ, Johnson JW, Bland DE, Kim DY, Seo YW. 2009. Development and functional assessment of EST-derived 2RL-specific markers for 2BS.2RL translocations. Theor Appl Genet, 119:663-673).
Yet up to the present, also nobody's report derives from the complete rye 1R～special molecule marker of 7R karyomit(e) of a cover of rye expressed sequence tag (EST) sequence, to be used for detecting the whole chromosomess of rye of identifying in the Wheat Background of 1R～7R.
Summary of the invention
The technical problem to be solved in the present invention provides the rye 1R～7R karyomit(e) special molecule marker of a cover based on the rye est sequence, designed and developed out the specific molecular marker of a cover chromosomes of rye based on the est sequence on the chromosomes of rye, can enough regular-PCR technology identify whole chromosomess of rye of 1R～7R in the wheat genetic background fast and accurately by these molecule markers, transferring to for desirable genes on the chromosomes of rye provides simple and effective molecular assay method in the cultivated wheat background.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Rye 1R～special molecule marker of 7R karyomit(e) based on the rye est sequence, forward primer and the reverse primer of these molecule markers for designing according to the est sequence on the chromosomes of rye, these molecule markers are identified the chromosomal specific mark of rye 1R～7R for being used to, and its base sequence sees Table 1
Table 1. is according to 14 pairs of labeled primer sequences of the design of the est sequence on the chromosomes of rye
Wherein, the 1-CGG143 mark is used to identify rye 1R and 6R karyomit(e); 2-CGG62,3-CGG8 and 4-CGG9 mark are used to identify rye 2R karyomit(e); The 5-CGG32 mark is used to identify rye 3R karyomit(e); The 6-CGG49 mark is used to identify rye 4R karyomit(e); 7-CGG4,8-CGG16,9-CGG18,10-CGG19 and 11-CGG43 mark are used to identify rye 5R karyomit(e); 12-CGG23 and 13-CGG59 mark are used to identify rye 6R karyomit(e); The 14-CGG26 mark is used to identify rye 7R karyomit(e).Wherein, " EST " is rows of has gone out the title of the est sequence on the pairing chromosomes of rye of molecule marker.
Use the above-mentioned method of chromosomes of rye in the Wheat Background being identified based on the special molecule marker of the rye 1R～7R karyomit(e) of rye est sequence, its step comprises:
Selected and the preparation of A, labeled primer: according to the selected molecule marker of the chromosomes of rye of required evaluation, such as, whether contain rye 4R karyomit(e) in the middle of preliminary detection wheat and the rye distant hybrid progeny material, then selected 6-CGG49 mark, synthetic this labeled primer of sequence that provides according to table 1 then;
The dilution of B, labeled primer: will go up step synthetic labeled primer, and be dissolved into 100mmol L with 1 * TE damping fluid earlier
-1Mother liquor put into-20 ℃ refrigerator and preserve, draw mother liquor before using and be diluted to 10mmol L with ultrapure water
-1Working fluid stand-by; Wherein the prescription of 1 * TE damping fluid is, 10mmol L
-1Tris-HCl and 1mmol L
-1EDTA, pH=8.0;
The specific mark of C, chromosomes of rye:
The distant hybrid progeny material of the extraction of C-1, template DNA: get determinand---wheat and rye, contrast 0---do not comprise the sample of chromosomes of rye to be measured, contrast 1---comprise the sample of chromosomes of rye to be measured, utilize conventional DNA extraction method to extract three's DNA respectively then;
C-2, pcr amplification: with C-1 gained DNA sample is template, utilizes step B gained working fluid respectively determinand, contrast 0 and contrast 1 to be increased in the pcr amplification instrument;
The detection of C-3, amplified production: native polyacrylamide gel electrophoresis detects step C-2 gained amplified production, dyes with argentation, and takes a picture on the gel imaging instrument;
C-4, result's comparison: the electrophoretogram of distant hybrid progeny material to be measured is compared with the electrophoretogram of contrast 0, contrast 1 respectively, if the electrophoretogram of distant hybrid progeny material to be measured and contrast 1 all contains the specific amplified fragment, illustrate and contain chromosomes of rye to be measured in the distant hybrid progeny material; If the electrophoretogram of distant hybrid progeny material to be measured is consistent with the electrophoretogram of contrast 0, all do not contain the specific amplified fragment, illustrate and do not contain chromosomes of rye to be measured in the distant hybrid progeny material.
As a kind of optimal technical scheme of aforesaid method, labeled primer selected in the steps A is synthetic by Shanghai associating gene company limited.
A kind of optimal technical scheme as aforesaid method, the concrete operations of among the step C-2 dna profiling of described determinand, contrast 0 and contrast 1 being increased are, the template DNA that in volume is the Eppendof pipe of 0.2ml, adds 30ng successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, each 0.2mmol L of forward and reverse primer
-1, 0.5U
TaqArchaeal dna polymerase uses aseptic ultrapure water postreaction system to 10ml at last, the Eppendof pipe is placed on carries out amplified reaction on the GeneAmp9700 gene-amplificative instrament then.
As a kind of optimal technical scheme of aforesaid method, the specific procedure of described amplified reaction is 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, according to the annealing temperature annealing 1min of labeled primer, 72 ℃ are extended 1min, circulate 38 times; Last 72 ℃ are extended 10min, 10 ℃ of preservations; Wherein the annealing temperature of each labeled primer is, 1-CGG143,50 ℃; 2-CGG62,60 ℃; 3-CGG8,62 ℃; 4-CGG9,56 ℃; 5-CGG32,55 ℃; 6-CGG49,55 ℃; 7-CGG4,60 ℃; 8-CGG16,57 ℃; 9-CGG18,56 ℃; 10-CGG19,58 ℃; 11-CGG43,52 ℃; 12-CGG23,62 ℃; 13-CGG59,51 ℃; 14-CGG26,60 ℃.
A kind of optimal technical scheme as aforesaid method, the detection method of amplified production described in the step C-3 is specially, in the amplified production of step C-2 gained, add sample-loading buffer 2 μ l, get 3 μ l after the mixing, detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, electrophoresis 1～2h.
Adopt the beneficial effect that technique scheme produced to be:
1. the specific molecular marker of 14 chromosomess of rye of the present invention exploitation is the mark of sequence convertibility, and the RAPD mark more used than forefathers is stable more and have a repeatability; The binding ability of labeled primer and template is stronger, can amplify target fragment well, and requirement for experiment condition is looser; Divide band to compare with the authentication method of in situ hybridization with the C-that identifies exogenous chromosome, operation steps is simple, and experimental technique requires lower, can identify the chromosomes of rye in the Wheat Background simply, apace;
2. 14 chromosomes of rye specific molecular markers that come from the rye est sequence of the present invention exploitation, covered whole rye 1R～7R karyomit(e), can detect and identify the whole chromosomess of rye of 1R～7R in the Wheat Background, provide the basis of molecular marker assisted selection for utilizing rye desirable genes improvement wheat;
3. the special mark of introducing in the present invention and the front document of chromosomes of rye based on the exploitation of wheat est sequence is compared, and the chromosomes of rye specific molecular marker of the present invention's exploitation is the est sequence that derives from rye, and its conservative property is stronger;
4. the present invention is according to the molecule marker of the est sequence exploitation of rye, has good transferability between rye and wheat, and this provides abundant mark source for saturated wheat genetic linkage map;
5. the molecule marker of the present invention exploitation, its sequence is directed to the gene transcription zone, and this lays a good foundation for the evaluation of rye functional gene.
Description of drawings
Fig. 1: the detected result of rye 5R karyomit(e) specific molecular marker 8-CGG16 in kingdom rye, common wheat China spring and " China spring-kingdom rye " disomic addition line (1R-7R); Wherein, M:marker pUC19/
MspI, arrow are shown in the specific amplification fragment in kingdom rye and the 5R addition line; 1: China spring, 2: kingdom rye, 3:1R addition line, 4:2R addition line, 5:3R addition line, 6:4R addition line, 7:5R addition line, 8:6R addition line, 9:7R addition line.
Fig. 2: rye 1R and the 6R karyomit(e) specific molecular marker 1-CGG143 detected result in kingdom rye, common wheat China spring and " China spring-kingdom rye " disomic addition line (1R-7R); Wherein, M:marker pUC19/
MspI, arrow are shown in the specific amplification fragment in kingdom rye and 1R, the 6R addition line; 1: China spring, 2: kingdom rye, 3:1R addition line, 4:2R addition line, 5:3R addition line, 6:4R addition line, 7:5R addition line, 8:6R addition line, 9:7R addition line.
Fig. 3: rye 1R and 6R karyomit(e) specific molecular marker 1-CGG143 common wheat " lay down for a short time No. 6 " and material such as " Germany white " rye filial generation in detected result; Wherein, M:marker pUC19/
MspI, arrow show the specific amplification fragment; 1: China spring, 2: the kingdom rye, 3: lay down for a short time No. 6,4: the white rye of Germany, the 5:WR64(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the 6:WR81(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the 7:WR91(white 2R(2D of No. 6-Germany that lays down for a short time) substitution line), the 8:WR41(No. 6-Germany white 4B/4R reciprocal translocation system of laying down for a short time), 9: the 1BL/1RS translocation line of Luo Fulin 10(rye Petkus), 10: the 1BL/1RS translocation line of Luo Fulin 13(rye Petkus), 11: triticale 06CT456(AABBRR), 12: triticale 06CT461(AABBRR).
Fig. 4: the detected result of rye 2R karyomit(e) specific molecular marker 2-CGG62 in 7 rye kinds, 4 wheat sibling specieses and 2 common wheats; Wherein, M:marker pUC19/
MspI, arrow show the specific amplification fragment; 1: China spring, 2: kingdom rye, 3: lay down 4 for a short time No. 6: the white rye of Germany, 5: Guyuan rye, 6:King rye, 7: U.S. rye, 8: Jing Zhou rye, 9: Austrian rye, 10: wheatgrass, 11: barley, 12: cluster hair wheat, 13: couchgrass.
Embodiment
Following examples describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercially available prod, all can buy directly by market to obtain.
Embodiment 1: the exploitation of rye 5R karyomit(e) specific molecular marker
Use the method that the 8-CGG16 molecule marker is identified rye 5R karyomit(e), detected materials is the chromosomal disome alien addition line of 5R of " China spring-kingdom rye ", and control material is kingdom rye, common wheat China spring and all the other 6 " China spring-kingdom rye " disome alien addition lines; Wherein, " China spring-kingdom rye " disome alien addition line (1R-7R) is a public material (reference: Driscoll C J, Sears E R, 1971. Individual additions of the chromosomes of " Imperial " rye to wheat. Agron Abstr, 1971:6); Operation steps is as follows:
The preparation of A, labeled primer: the sequence according to the 8-CGG16 labeled primer is synthetic by Shanghai associating gene company limited;
The dilution of B, labeled primer: will go up step synthetic labeled primer, and be dissolved into 100mmol L with 1 * TE damping fluid earlier
-1Mother liquor put into-20 ℃ refrigerator and preserve, draw mother liquor before using and be diluted to 10mmol L with ultrapure water
-1Working fluid stand-by; Wherein the prescription of 1 * TE damping fluid is, 10mmol L
-1Tris-HCl and 1mmol L
-1EDTA, pH=8.0;
The specific mark of C, chromosomes of rye:
The extraction of C-1, template DNA: get the chromosomal disome alien addition line of 5R of detected materials " China spring-kingdom rye " and control material kingdom rye, common wheat China spring, all the other 6 " China spring-kingdom rye " disome alien addition lines, utilize conventional DNA extraction method to extract its DNA respectively;
C-2, pcr amplification: with C-1 gained DNA sample is template, utilize step B gained working fluid in the pcr amplification instrument respectively the chromosomal disome alien addition line of 5R, kingdom rye, common wheat China spring, all the other 6 " China spring-kingdom rye " the disome alien addition lines to " China spring-kingdom rye " increase; The concrete operations of amplification experiment are, the Eppendof pipe that to get 9 volumes be 0.2ml is to the template DNA that wherein adds 30ng successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, each 0.2mmol L of forward and reverse primer
-1, 0.5U
TaqArchaeal dna polymerase uses aseptic ultrapure water postreaction system to 10ml at last, the Eppendof pipe is placed on carries out amplified reaction on the GeneAmp9700 gene-amplificative instrament then; The specific procedure of amplified reaction is 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, according to the annealing temperature annealing 1min of labeled primer, 72 ℃ are extended 1min, circulate 38 times; Last 72 ℃ are extended 10min, 10 ℃ of preservations; Wherein the annealing temperature of 8-CGG16 labeled primer is 57 ℃; Finally obtain the chromosomal disome alien addition line of 5R, kingdom rye, common wheat China spring, all the other 6 " China spring-kingdom rye " the disome alien addition line amplified productions separately of " China spring-kingdom rye ", amount to 9 parts;
The detection of C-3, amplified production: in 9 parts of amplified productions of step C-2 gained, add sample-loading buffer 2 μ l respectively, get 3 μ l after the mixing, detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, electrophoresis 1.5h, so dye, and on the gel imaging instrument, take a picture with argentation;
C-4, result's comparison: referring to accompanying drawing 1, among the figure: M:marker pUC19/
MspI, arrow are shown in the specific amplification fragment in kingdom rye and the 5R addition line; 1: China spring, 2: kingdom rye, 3:1R addition line, 4:2R addition line, 5:3R addition line, 6:4R addition line, 7:5R addition line, 8:6R addition line, 9:7R addition line; 8-CGG16 all has the specific amplification of about 250bp in the chromosomal disome alien addition line of 5R of kingdom rye and " China spring-kingdom rye ", and at equal this specific amplification not of China spring and all the other 6 " China spring-kingdom rye " disome alien addition lines; This shows that 8-CGG16 is the chromosomal specific molecular marker of rye 5R, can be used to rye 5R karyomit(e) is carried out quick, easy special tracking and evaluation.
Embodiment 2: the exploitation of rye 1R and 6R karyomit(e) specific molecular marker
Use the method that the 1-CGG143 mark is identified rye 1R and 6R karyomit(e), detected materials is the 1R and the chromosomal disome alien addition line of 6R of " China spring-kingdom rye ", and control material is kingdom rye, common wheat China spring and all the other 5 " China spring-kingdom rye " disome alien addition lines; Operation steps is as follows:
The preparation of A, labeled primer: the sequence according to the 1-CGG143 labeled primer is synthetic by Shanghai associating gene company limited;
The dilution of B, labeled primer: will go up step synthetic labeled primer, and be dissolved into 100mmol L with 1 * TE damping fluid earlier
-1Mother liquor put into-20 ℃ refrigerator and preserve, draw mother liquor before using and be diluted to 10mmol L with ultrapure water
-1Working fluid stand-by; Wherein the prescription of 1 * TE damping fluid is, 10mmol L
-1Tris-HCl and 1mmol L
-1EDTA, pH=8.0;
The specific mark of C, chromosomes of rye:
The extraction of C-1, template DNA: get the 1R of detected materials " China spring-kingdom rye " and the chromosomal disome alien addition line of 6R and control material kingdom rye, common wheat China spring, all the other 5 " China spring-kingdom rye " disome alien addition lines; Utilize conventional DNA extraction method to extract its DNA respectively;
C-2, pcr amplification: with C-1 gained DNA sample is template, utilize step B gained working fluid in the pcr amplification instrument respectively 1R and the chromosomal disome alien addition line of 6R, kingdom rye, common wheat China spring, all the other 5 " China spring-kingdom rye " the disome alien addition lines to " China spring-kingdom rye " increase; The concrete operations of amplification experiment are, the Eppendof pipe that to get 9 volumes be 0.2ml is to the template DNA that wherein adds 30ng successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, each 0.2mmol L of forward and reverse primer
-1, 0.5U
TaqArchaeal dna polymerase uses aseptic ultrapure water postreaction system to 10ml at last, the Eppendof pipe is placed on carries out amplified reaction on the GeneAmp9700 gene-amplificative instrament then; The specific procedure of amplified reaction is 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, according to the annealing temperature annealing 1min of labeled primer, 72 ℃ are extended 1min, circulate 38 times; Last 72 ℃ are extended 10min, 10 ℃ of preservations; Wherein the annealing temperature of 1-CGG143 labeled primer is 50 ℃; Finally obtain the 1R of " China spring-kingdom rye " and the chromosomal disome alien addition line of 6R, kingdom rye, common wheat China spring, all the other 5 " China spring-kingdom rye " disome alien addition line amplified productions separately, amount to 9 parts;
The detection of C-3, amplified production: in 9 parts of amplified productions of step C-2 gained, add sample-loading buffer 2 μ l respectively, get 3 μ l after the mixing, detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, electrophoresis 1.5h, so dye, and on the gel imaging instrument, take a picture with argentation;
C-4, result's comparison: referring to accompanying drawing 2, among the figure: M:marker pUC19/
MspI, arrow are shown in the specific amplification fragment in kingdom rye and 1R, the 6R addition line; 1: China spring, 2: kingdom rye, 3:1R addition line, 4:2R addition line, 5:3R addition line, 6:4R addition line, 7:5R addition line, 8:6R addition line, 9:7R addition line; Mark 1-CGG143 has the specific amplification of about 150bp and 100bp respectively in the 1R of kingdom rye and " China spring-kingdom rye " and the chromosomal disome alien addition line of 6R, and in China spring and all the other 5 " China spring-kingdom rye " disome alien addition lines equal this specific amplifications not; This shows the amplification site that has mark 1-CGG143 on rye 1R and the 6R karyomit(e) respectively, can utilize 1-CGG143 that rye 1R and 6R karyomit(e) are carried out quick, easy tracking and evaluation.
Embodiment 3: the application of rye 1R and 6R karyomit(e) specific molecular marker
Use that the 1-CGG143 mark " is laid down No. 6 " for a short time to common wheat and material such as " Germany in vain " rye filial generation in the method identified of rye 1R and 6R karyomit(e), detected materials is the white rye of Germany, the WR64(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the WR81(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the WR91(white 2R(2D of No. 6-Germany that lays down for a short time) the WR41(No. 6-Germany white 4B/4R reciprocal translocation system of laying down for a short time substitution line)); Control material is a China spring, lays down kingdom rye, the 1BL/1RS translocation line of Luo Fulin 10(Petkus rye for a short time No. 6), the 1BL/1RS translocation line of Luo Fulin 13(Petkus rye), triticale 06CT456(AABBRR) and, triticale 06CT461(AABBRR); Operation steps is as follows:
The preparation of A, labeled primer: the sequence according to the 1-CGG143 labeled primer is synthetic by Shanghai associating gene company limited;
The dilution of B, labeled primer: will go up step synthetic labeled primer, and be dissolved into 100mmol L with 1 * TE damping fluid earlier
-1Mother liquor put into-20 ℃ refrigerator and preserve, draw mother liquor before using and be diluted to 10mmol L with ultrapure water
-1Working fluid stand-by; Wherein the prescription of 1 * TE damping fluid is, 10mmol L
-1Tris-HCl and 1mmol L
-1EDTA, pH=8.0;
The specific mark of C, chromosomes of rye:
The extraction of C-1, template DNA: get detected materials, utilize conventional DNA extraction method to extract its DNA respectively, amount to 12 parts;
C-2, pcr amplification: with C-1 gained DNA sample is template, utilizes step B gained working fluid in the pcr amplification instrument 12 parts of testing samples to be increased respectively; The concrete operations of amplification experiment are, the Eppendof pipe that to get 12 volumes be 0.2ml is to the template DNA that wherein adds 30ng successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, each 0.2mmol L of forward and reverse primer
-1, 0.5U
TaqArchaeal dna polymerase uses aseptic ultrapure water postreaction system to 10ml at last, the Eppendof pipe is placed on carries out amplified reaction on the GeneAmp9700 gene-amplificative instrament then; The specific procedure of amplified reaction is 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, according to the annealing temperature annealing 1min of labeled primer, 72 ℃ are extended 1min, circulate 38 times; Last 72 ℃ are extended 10min, 10 ℃ of preservations; Wherein the annealing temperature of 8-CGG16 labeled primer is 57 ℃; Finally obtain China spring, the kingdom rye, for a short time lay down No. 6, the white rye of Germany, the WR64(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the WR81(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the WR91(white 2R(2D of No. 6-Germany that lays down for a short time) substitution line), the WR41(No. 6-Germany white 4B/4R reciprocal translocation system of laying down for a short time), the 1BL/1RS translocation line of Luo Fulin 10(Petkus rye), the 1BL/1RS translocation line of Luo Fulin 13(Petkus rye), triticale 06CT456(AABBRR), triticale 06CT461(AABBRR) amplified production separately amounts to 12 parts;
The detection of C-3, amplified production: in 12 parts of amplified productions of step C-2 gained, add sample-loading buffer 2 μ l respectively, get 3 μ l after the mixing, detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, electrophoresis 1.5h, so dye, and on the gel imaging instrument, take a picture with argentation;
C-4, result's comparison: referring to accompanying drawing 3, among the figure: M:marker pUC19/
MspI, arrow show the specific amplification fragment; 1: China spring, 2: the kingdom rye, 3: lay down for a short time No. 6,4: the white rye of Germany, the 5:WR64(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the 6:WR81(white 1BL/1RS translocation line of No. 6-Germany of laying down for a short time), the 7:WR91(white 2R(2D of No. 6-Germany that lays down for a short time) substitution line), the 8:WR41(No. 6-Germany white 4B/4R reciprocal translocation system of laying down for a short time), 9: the 1BL/1RS translocation line of Luo Fulin 10(Petkus rye), 10: the 1BL/1RS translocation line of Luo Fulin 13(Petkus rye), 11: triticale 06CT456(AABBRR), 12: triticale 06CT461(AABBRR); 1-CGG143 is marked with two amplification sites, lay respectively on rye 1R and the 6R karyomit(e), at its site on rye 1R karyomit(e), it all amplifies the specific fragment of about 150bp in kingdom rye, the white rye of Germany, triticale 06CT456 and 06CT461, simultaneously in WR64, WR81, Luo Fulin 10 and Luo Fulin 13 these 4 1BL/1RS translocation lines, all amplify the polymorphism specific fragment that is slightly less than 150bp, all do not occur this specific amplified fragment in the chromosomal material of 1R and do not carry at other; Be marked at site on the rye 6R karyomit(e) at 1-CGG143, it only amplifies the specific fragment of about 100bp in kingdom rye, the white rye of Germany, triticale 06CT456 and 06CT461, and all do not carry and all do not occur this specific amplified fragment in the chromosomal material of 6R at other.Qualification result is consistent with the result of embodiment 2, illustrates that the 1-CGG143 mark can be used for detecting the 1R that identifies in the Wheat Background and the chromosomes of rye of 6R well.
Embodiment 4: the checking of rye 2R karyomit(e) specific molecular marker
Use the 2-CGG62 mark rye 2R karyomit(e) of originating different is identified that detected materials comprises 7 rye kinds; Control material is 4 wheat sibling specieses and 2 common wheats; The former comprises: kingdom rye, the white rye of Germany, Guyuan rye, King rye, U.S. rye, Jing Zhou rye, Austrian rye; The latter comprises: wheatgrass, and barley, cluster hair wheat, couchgrass and China spring are laid down No. 6 for a short time, and operation steps is as follows:
The preparation of A, labeled primer: the sequence according to the 2-CGG62 labeled primer is synthetic by Shanghai associating gene company limited;
The dilution of B, labeled primer: will go up step synthetic labeled primer, and be dissolved into 100mmol L with 1 * TE damping fluid earlier
-1Mother liquor put into-20 ℃ refrigerator and preserve, draw mother liquor before using and be diluted to 10mmol L with ultrapure water
-1Working fluid stand-by; Wherein the prescription of 1 * TE damping fluid is, 10mmol L
-1Tris-HCl and 1mmol L
-1EDTA, pH=8.0;
The specific mark of C, chromosomes of rye:
The extraction of C-1, template DNA: get detected materials, utilize conventional DNA extraction method to extract its DNA respectively, amount to 13 parts;
C-2, pcr amplification: with C-1 gained DNA sample is template, utilizes step B gained working fluid in the pcr amplification instrument 13 parts of testing samples to be increased respectively; The concrete operations of amplification experiment are, the Eppendof pipe that to get 13 volumes be 0.2ml is to the template DNA that wherein adds 30ng successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, each 0.2mmol L of labeled primer
-1, 0.5U
TaqArchaeal dna polymerase uses aseptic ultrapure water postreaction system to 10ml at last, the Eppendof pipe is placed on carries out amplified reaction on the GeneAmp9700 gene-amplificative instrament then; The specific procedure of amplified reaction is 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, according to the annealing temperature annealing 1min of labeled primer, 72 ℃ are extended 1min, circulate 38 times; Last 72 ℃ are extended 10min, 10 ℃ of preservations; Wherein the annealing temperature of 8-CGG16 labeled primer is 57 ℃; Finally obtain 7 rye kinds---kingdom rye, the white rye of Germany, Guyuan rye, King rye, U.S. rye, Jing Zhou rye, Austrian rye; 4 wheat sibling specieses---wheatgrass, barley, cluster hair wheat, couchgrass and 2 common wheats---China spring are laid down No. 6 for a short time, and amplified production separately amounts to 13 parts;
The detection of C-3, amplified production: in 13 parts of amplified productions of step C-2 gained, add sample-loading buffer 2 μ l respectively, get 3 μ l after the mixing, detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, electrophoresis 1.5h, so dye, and on the gel imaging instrument, take a picture with argentation;
C-4, result's comparison: referring to accompanying drawing 4, among the figure: M:marker pUC19/
MspI, arrow show the specific amplification fragment; 1: China spring, 2: kingdom rye, 3: lay down 4 for a short time No. 6: the white rye of Germany, 5: Guyuan rye, 6:King rye, 7: U.S. rye, 8: Jing Zhou rye, 9: Austrian rye, 10: wheatgrass, 11: barley, 12: cluster hair wheat, 13: couchgrass; 2-CGG62 is in 7 rye kinds---kingdom rye, and Germany is rye in vain, the Guyuan rye, the King rye, U.S. rye, Jing Zhou rye, in the Austria rye specific amplification of about 250bp is arranged all, and, lay down for a short time No. 6 in 2 wheats and 4 sibling specieses---China spring thereof; Wheatgrass, barley, cluster hair wheat, equal this specific amplification not in the couchgrass.Illustrate that using the 2-CGG62 mark can accurately identify the rye 2R karyomit(e) of originating different well.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the single restricted condition to its technical scheme itself.
Sequence table
SEQ?ID?NO?1:
TCGGATCACA?TTCACTTA?18
SEQ?ID?NO?2:
TGGCGTCTTG?ACTACATT?18
SEQ?ID?NO?3:
GCCCTCGACG?ACATGAAA?18
SEQ?ID?NO?4:
CGCTTGCCGG?TCTTGTAT?18
SEQ?ID?NO?5:
ATCCATCCCA?TCCCGTCTC?19
SEQ?ID?NO?6:
TGGCTACACT?CGCTCTCGTC?20
SEQ?ID?NO?7:
CAGAGCAACA?GCGACATCTT?C?21
SEQ?ID?NO?8:
TCAACCCAAG?GCAAAAGG?18
SEQ?ID?NO?9:
GGAGGTGAGA?CAACAAGAC?19
SEQ?ID?NO?10:
CAGACGGCAA?TGTGATAG?18
SEQ?ID?NO?11:
GAACGCAAGC?ACTTCTCA?18
SEQ?ID?NO?12:
GCTCCTTTCT?AAGCCTGTC?19
SEQ?ID?NO?13:
CGAGGTGAAC?GATGATGACA?GC?22
SEQ?ID?NO?14:
TAGCAATGAG?CAGCAACGGC?20
SEQ?ID?NO?15:
ACCGTTCATC?CATCGTTCC?19
SEQ?ID?NO?16:
ACCAACCAAT?GTTGCTCCC?19
SEQ?ID?NO?17:
ACACTCCAGG?GTATGACACA?GG?22
SEQ?ID?NO?18:
CGGTTTAGAT?GCGTCGTTG?19
SEQ?ID?NO?19:
ATGCGTGCCT?CTTTGATGC?19
SEQ?ID?NO?20:
CGGATGTGTG?GATGGTTTTG?20
SEQ?ID?NO?21:
GGAGCAACCA?TCTTGAGTA?19
SEQ?ID?NO?22:
TGTCTGGAAG?GACCGTAG?18
SEQ?ID?NO?23:
TCGTCTCGCA?GACCTTGCAC?20
SEQ?ID?NO?24:
CAGCAACGCA?TCGACTGAGC?20
SEQ?ID?NO?25:
AGCTACGTGG?AGCACAAG?18
SEQ?ID?NO?26:
TGATGATACG?CACAACAAA?19
SEQ?ID?NO?27:
GCTGGTTGGA?ATAACGCTGA?TG?22
SEQ?ID?NO?28:
CGGCAAGCAA?TGAGAGACAG?AG?22
Claims (6)
1. based on the rye 1R～special molecule marker of 7R karyomit(e) of rye est sequence, it is characterized in that: these molecule markers are forward primer and reverse primer according to the design of the expressed sequence tag est sequence on the chromosomes of rye, these molecule markers are for being used to identify the chromosomal specific mark of rye 1R～7R, its base sequence sees Table 1
Table 1. is according to 14 pairs of labeled primer sequences of the design of the expressed sequence tag est sequence on the chromosomes of rye
Wherein, the 1-CGG143 mark is used to identify rye 1R and 6R karyomit(e); 2-CGG62,3-CGG8 and 4-CGG9 mark are used to identify rye 2R karyomit(e); The 5-CGG32 mark is used to identify rye 3R karyomit(e); The 6-CGG49 mark is used to identify rye 4R karyomit(e); 7-CGG4,8-CGG16,9-CGG18,10-CGG19 and 11-CGG43 mark are used to identify rye 5R karyomit(e); 12-CGG23 and 13-CGG59 mark are used to identify rye 6R karyomit(e); The 14-CGG26 mark is used to identify rye 7R karyomit(e).
2. use the claim 1 described method of chromosomes of rye in the Wheat Background being identified based on the special molecule marker of the rye 1R～7R karyomit(e) of rye est sequence, its characterization step comprises:
Selected and the preparation of A, molecule marker: according to the selected molecule marker of the chromosomes of rye of required evaluation, such as, whether contain rye 4R karyomit(e) in the middle of wheat that preliminary detection is identified and the rye distant hybrid progeny material, then selected 6-CGG49 molecule marker, synthetic this labeled primer of sequence that provides according to claim 1 then;
The dilution of B, labeled primer: will go up step synthetic labeled primer, and be dissolved into 100mmol L with 1 * TE damping fluid earlier
-1Mother liquor put into-20 ℃ refrigerator and preserve, draw mother liquor before using and be diluted to 10mmol L with ultrapure water
-1Working fluid stand-by; Wherein the prescription of 1 * TE damping fluid is, 10mmol L
-1Tris-HCl and 1mmol L
-1EDTA, pH=8.0;
The specific mark of C, chromosomes of rye:
The extraction of C-1, template DNA: get determinand---wheat and rye distant hybrid progeny material, contrast 0---do not comprise the sample of chromosomes of rye to be measured, contrast 1---comprise the sample of chromosomes of rye to be measured, utilize conventional DNA extraction method to extract three's DNA respectively then;
C-2, pcr amplification: with C-1 gained DNA sample is template, utilizes step B gained working fluid respectively determinand, contrast 0 and contrast 1 to be increased in the pcr amplification instrument;
The detection of C-3, amplified production: native polyacrylamide gel electrophoresis detects the amplified production of step C-2 gained, dyes with argentation, and takes a picture on the gel imaging instrument;
C-4, result's comparison: the electrophoretogram of distant hybrid progeny material to be measured is compared with the electrophoretogram of contrast 0, contrast 1 respectively, if the electrophoretogram of distant hybrid progeny material to be measured and contrast 1 all has the specific amplified fragment, illustrate and contain chromosomes of rye to be measured in the distant hybrid progeny material; If the electrophoretogram of distant hybrid progeny material to be measured is consistent with the electrophoretogram of contrast 0, all do not contain the specific amplified fragment, illustrate and do not contain chromosomes of rye to be measured in the distant hybrid progeny material.
3. according to the claim 2 described methods that chromosomes of rye is identified, it is characterized in that: labeled primer selected in the steps A is synthetic by Shanghai associating gene company limited.
4. according to the claim 2 described methods that chromosomes of rye is identified, it is characterized in that: the concrete operations of among the step C-2 dna profiling of described determinand, contrast 0 and contrast 1 being increased are, the template DNA that in volume is the Eppendof pipe of 0.2ml, adds 30ng successively, 1 * PCR buffer, 1.5mmol L
-1MgCl
2, 200mmol L
-1DNTP, each 0.2mmol L of forward and reverse primer
-1, 0.5U
TaqArchaeal dna polymerase uses aseptic ultrapure water postreaction system to 10ml at last, the Eppendof pipe is placed on carries out amplified reaction on the GeneAmp9700 gene-amplificative instrament then.
5. according to the claim 4 described methods that chromosomes of rye is identified, it is characterized in that: the specific procedure of described amplified reaction is 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of sex change 1min then, according to the annealing temperature annealing 1min of labeled primer, 72 ℃ are extended 1min, circulate 38 times; Last 72 ℃ are extended 10min, 10 ℃ of preservations; Wherein the annealing temperature of each labeled primer is, 1-CGG143,50 ℃; 2-CGG62,60 ℃; 3-CGG8,62 ℃; 4-CGG9,56 ℃; 5-CGG32,55 ℃; 6-CGG49,55 ℃; 7-CGG4,60 ℃; 8-CGG16,57 ℃; 9-CGG18,56 ℃; 10-CGG19,58 ℃; 11-CGG43,52 ℃; 12-CGG23,62 ℃; 13-CGG59,51 ℃; 14-CGG26,60 ℃.
6. according to the claim 2 described methods that chromosomes of rye is identified, it is characterized in that: the detection method of amplified production described in the step C-3 is specially, in the amplified production of step C-2 gained, add sample-loading buffer 2 μ l, get 3 μ l after the mixing, detect with 8% native polyacrylamide gel electrophoresis, electrophoretic voltage is 150～180V, electrophoresis 1～2h.
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| CN201210479388.9A Division CN102925439B (en) | 2011-04-14 | 2011-04-14 | Secale cereale EST (expressed sequence tag) sequence based specific molecular marker of secale cereale 2R chromosome and application thereof |
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| CN109234449A (en) * | 2018-11-29 | 2019-01-18 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | A kind of special codominance KASP molecular labeling of the general 2RL chromosome of rye and its application |
| CN109280665A (en) * | 2018-11-06 | 2019-01-29 | 西北农林科技大学 | Rye molecular specificity labeled primers, application method and its application |
| CN111235292A (en) * | 2020-02-21 | 2020-06-05 | 烟台大学 | Rye 4RS chromosome arm specific KASP molecular marker and application thereof |
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| CN109234449B (en) * | 2018-11-29 | 2022-04-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Universal 2RL chromosome specific codominant KASP molecular marker for rye and application thereof |
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| CN111235292B (en) * | 2020-02-21 | 2022-09-27 | 烟台大学 | Rye 4RS chromosome arm specific KASP molecular marker and application thereof |
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