CN102703445A - Specific molecular markers of eIF4E.a mutation site of Chinese cabbage and application of Specific molecular markers - Google Patents
Specific molecular markers of eIF4E.a mutation site of Chinese cabbage and application of Specific molecular markers Download PDFInfo
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Abstract
The invention discloses two site-specific dominant ASM markers which are directly related with the identification of a wild type and a mutant of eIF4E.a of Chinese cabbage. One marker is that: a marker which is related with the detection of a wild type site is named ASM-4E.a-s and is shown as SEQ ID No.1; and a marker which is related with the detection of the corresponding mutation site is named ASM-4e.a-s and is shown as SEQ ID No.3. The other marker is that: a marker which is related with the detection of a wild type site is named ASM-4E.a-lc and is shown as SEQ ID No.2; and a marker which is related with the detection of the corresponding mutation site is named ASM-4e.a-1c and is shown as SEQ ID No.4. The invention also discloses a primer for identifying the two specific dominant molecular markers. The primer is shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7. The markers and the primer can be taken as molecular markers and applied to the identification of germplasm resources of the Chinese cabbage and the aided selection of breeding; and a germplasm material or a transformation offspring containing an eIF4E.a mutation type in 06-247 or Guan291 is selected.
Description
Technical field
The present invention relates to a kind of exploitation of two mutants and related locus specific molecular marker thereof of gene, relate in particular to sudden change and locus specificity molecular markers development and the application of a kind of Chinese cabbage eukaryotic translation initiator eIF4E.a.Two mutants can provide the variation source for the antiviral Chinese cabbage germplasm that seed selection contains this mutational site; The site-specific molecule marker can directly apply to the Chinese cabbage material that molecular mark selects to contain this mutational site; Improve the efficiency of selection of eIF4E two mutants, belong to biological technical field.
Background technology
Chinese cabbage (Brassica rapa L.ssp pekinensis) is the important vegetable crop of Cruciferae, originates in China, and plantation is all arranged all over the world at present.Especially in the year-round provisions of China vegetables and s are adjusted, become the important component part that vegetable prods is supplied with, thereby people's life has been had material impact.In the Chinese cabbage production process, often suffer the threat of diseases such as virus disease, oidium and soft rot.The harm of virus disease is the most serious as a complete unit, and does not have blanket viral diseases medicament or other effective control techniques.Cultivate antiviral Chinese cabbage new variety and be the harm of reply virus disease first-selected approach [Wang Xue, Liu Yumei, Li Hanxia, make widely known brave, Fang Zhiyuan. rape belongs to crop resisting turnip mosaic virus Research Advances on Breeding. gardening journal .2005,32 (5): 939-946].Upright strong [the Miao Liqiang that waits of seedling; Zhang Yaowei; Cui Chongshi. China's Chinese cabbage breeding for disease resistance progress. the journal .2008 of Northeast Agricultural University, 37 (4): 529-533] discover that 70% is Brassica 2 et 4 (Turnip Mosaic Virus in the pathogen separation thing of China's Chinese cabbage virus disease; TuMV), (Cucumber Mosaic Virus is CMV) with other virus to also have a small amount of cucumber mosaic virus.Therefore the main goal of attack of China's Chinese cabbage viral diseases breeding is anti-TuMV.
Two key elements cultivating anti-TuMV Chinese cabbage new variety are to have abundant anti-TuMV germ plasm resource and breeding efficiency efficiently.China is the Chinese cabbage country of origin, and germ plasm resource is very abundant, wherein is no lack of the good resource of anti-TuMV, and more how best in quality but not antiviral resource is also arranged simultaneously.In the conventional breeding practice, often the means through backcross transformation obtain the abundant resistant material of more genetic backgrounds.Along with the rise of molecular marking technique and going deep into of research, marker assisted selection has become in the conventional breeding operation and has improved efficiency of selection, the main means of shortening the breeding cycle.Some transnational breeding groups spare no high price innovation resources and exploitation and the closely linked molecule marker of the Main Agronomic Characters main contents as the breeding tackling key problem especially.
Generally speaking, seek and the closely linked molecule marker of certain economical character, often need be chosen in the parent that there is significant difference in the objective trait aspect earlier, and make up segregating population (F2; RI, BC1, DH etc.); Adopt to mix hive off (BSA) and traditional molecule marker (like RAPD, SRAP, AFLP; SSR etc.) technology is analyzed, and the linkage distance with corresponding software analysis gained mark and proterties finally obtains the molecule marker chain with objective trait.The molecule marker with Chinese cabbage resisting turnip mosaic virus (TuMV) linkage of characters of report adopts aforesaid method to obtain just at present.Because different investigator's material therefors are different with the mark selected type, the chain marking path of resulting and ntiviral characteristic is also different, between 3.8-15.36cM.Because the linkage degree undertighten of these marks and proterties, thereby it is used for the anti-TuMV assistant breeding of Chinese cabbage certain distance is still arranged.
On the other hand, because Arabidopis thaliana and Chinese cabbage belong to Cruciferae together, its antiviral correlation function gene has more deep research; The whole genome sequence of Chinese cabbage is announced (Wang et al., 2011, the genome of the mesopolyploid crop species Brassica rapa.Nature Genetics.43 (10): 1035-1039) simultaneously.So can be with the result and the Chinese cabbage genome sequence column information of Arabidopis thaliana functional gene research; Directly start with from the angle of antiviral correlation function gene; Through Chinese cabbage natural population is started with in the natural variation of some critical function gene locuss, seek the two mutants of antiviral correlation function gene; To mutational site exploitation locus specificity molecule marker (Allele specific marker, ASM), then this mark itself is directly related with proterties, this can make marker assisted selection more accurate undoubtedly.
Discovered in recent years aspect antiviral functional gene research, how relevant by the antiviral proterties of recessive single-gene control with the sudden change of eukaryotic translation initiator 4E and isomer thereof.[Wittmann S such as Wittmann; Chatel H; Fortin MG; Laliberte JF.Interaction of the viral protein genome linked of Turnip mosaic virus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system.Virology.1997; 234:84-92] and [L é onard S, Plante D, Wittmann S such as L é onard; Daigneault N; Fortin MG, Lalibert é JF.Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity.J.Virol.2000,74:7730-7737] prove respectively: Arabidopis thaliana eIF4E and eIF (iso) 4E can interact with the viral genome of TuMV conjugated protein (VPg).The required key amino acid site of this interactional key amino acid and eukaryote self mRNA translation is different; So some amino acid mutations of eIF4E and eIF (iso) 4E do not influence grow, but cause virus and the host of plant self mutual work can not take place, thereby make the host show the characteristic of opposing virus infection.Ryder at romaine lettuce [Ryder EJ.1970.Inheritance of resistance to common lettuce mosaic.Am Soc Horticult Sci.95:378 – 379], Ruffel at capsicum [Ruffel S; Dussault MH; Palloix A; Moury B; Bendahmane A, Robaglia C, Caranta be natural recessive resistance gene against potato virus Y in peper corresponds to the eukaryotic initiation factor 4E (eIF4E) .Plant Dec J.2002 C.2002.A; 32 (6): 1067-75], Nieto is at muskmelon [Nieto C, Piron F, Dalmais M; Marco CF, Moriones E, Gomez-Guillamon ML; Truniger V, Gomez P, Garcia-Mas J; Aranda MA, Bendahmane be for the identification of allelic variants of melon eIF4E A.2007.EcoTILLING, a factor that controls virus susceptibility.BMC Plant Biology.7; 34-42] etc. important vegetables had been found that many such two mutants resources on; [Yeam I, Kang BC, Lindeman W such as Yeam; Frantz JD; Faber N, and Jahn MM.2005.Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum.Theor.Appl.Genet.NNO, 178-186] assisted Selection when in capsicum, having developed corresponding locus specificity CAPs mark and being used for antiviral material transformation to the mutational site.
China's Chinese cabbage germ plasm resource is very abundant; EIF4E and eIF (iso) 4E sudden change and the supposition of the effect antiviral thereof from other crops; The two mutants that also possibly have eIF4E and eIF (iso) 4E in the Chinese cabbage natural population, and this sudden change maybe be relevant with the ntiviral characteristic of Chinese cabbage.At present, the research that relevant both at home and abroad Chinese cabbage ntiviral characteristic and eIF4E and eIF (iso) 4E concern seldom, the material that relates to is very limited.[Rusholme RL such as Rusholme; Higgins EE; Walsh JA; Lydiate DJ.Genetic control of broad-spectrum resistance to turnip mosaic virus in Brassica rapa (Chinese cabbage) .Journal of General Virology.2007; 88:3177 – 3186] be the parent with Chinese cabbage inbred lines RLR22 and the viral sensitive material R-O-18 of the anti-TuMV of height, make up BC1F1 colony, carry out anti-TuMV (CDN 1 and CZE1 strain system) genetic research.The result has found the antiviral recessive inheritance of a Chinese cabbage site retr01 and a dominant locus ConTR01, and the association analysis of RFLP mark has been found respectively and chain mark pN202e1 of retr01 and the mark pO85e1 chain with ConTR01.Further adopt the means of southern hybridization to find that the eIF4E (laying respectively at karyomit(e) N1, N3 and N8) of 3 copies and eIF (iso) 4E (laying respectively at karyomit(e) N4, N5 and N8) of 3 copies are arranged respectively in the A genome.The author infer that the eIF4E that is positioned on the 8th karyomit(e) maybe be relevant with ConTR01 with eIF (iso) 4E, and eIF (iso) 4E that is positioned on the 4th karyomit(e) maybe be relevant with retr01 according to results of hybridization.Do not report in the literary composition about the sequence information of two genes.[Jenner CE such as Jenner; Nellist CF; Barker GC; Walsh JA.Turnip mosaic virus (TuMV) is able to use alleles of both eIF4E and eIF (iso) 4E from multiple loci of the diploid Brassica rapa.Mol Plant Microbe Interact.2010; 3 (11): 1498-1505] clone has obtained eIF4E and each three copy of eIF (iso) 4E, called after BraA.eIF4E.a, BraA.eIF4E.b, BraA.eIF4E.c and BraA.eIF (iso) 4E.a, BraA.eIF (iso) 4E.b, BraA.eIF (iso) 4E.c respectively from viral sensitive material R-O-18.Except that BraA.eIF4E.b and BraA.eIF (iso) the 4E.b; All the other 4 genes are arabidopsis thaliana transformation At.eIF (iso) 4E afunction two mutants [DupratA, Caranta C, Revers F respectively; Menand B; Browning KS, Robaglia be Arabidopsis eukaryotic initiation factor (iso) 4E is dispensable for plant growth but required for susceptibility to potyviruses.The Plant Journal C.2002.The, 32:927 – 934]; Be CDN1 to all transgenic line inoculation TuMV Canada strains then, carry out viral susceptibility complementation test.According to disease index and ELISA result, find that four genes that changeed all can make two mutants recover the susceptibility that TuMV is infected wholly or in part.This shows that when TuMV infected the Chinese cabbage group crop, eIF4E and eIF (iso) 4E possibly participate in virus and host's interaction.The author points out simultaneously, wants the acquisition antiviral material relevant with eIF (iso) 4E with eIF4E in Chinese cabbage, and then these several genes need loss of function simultaneously.Up to now, Chinese cabbage does not appear in the newspapers in the sudden change of above-mentioned two gene locuss and the marker development of related mutants detection both at home and abroad.
Given this, be necessary to the Chinese cabbage of China's abundant anti-/ sense TuMV material in the polymorphum in eIF4E and each site of eIF (iso) 4E carry out extensive examination, with the mutation type of finding that each site possibly exist; To the sequence difference of mutational site and wild-type, exploitation detects relevant locus specificity molecule marker with two mutants simultaneously.The potential benefit that the following aspects is arranged: 1. can this mark be used for examination Chinese cabbage population, Chinese cabbage two mutants detection means efficiently is provided; 2. when mutation type is found in other sites, can adopt the exploitation mark that uses the same method; 3. when a plurality of site mutation are arranged in material different, can many site mutation be condensed together, create the anti-TuMV Chinese cabbage of wide spectrum new germ plasm through the marker assisted selection means; 4. when cultivating the anti-TuMV Chinese cabbage of wide spectrum new variety, can realize marker assisted selection, improve breeding efficiency.Because this mark is located immediately at functional gene inside, the rate of accuracy reached of therefore selecting 100%.
Summary of the invention
To above-mentioned prior art; To present Chinese cabbage in the blank aspect eIF4E and the evaluation of eIF (iso) 4E gene point mutation body; The present invention has at first obtained two eIF4E.a site mutation bodies; Secondly according to the sequence characteristic in mutational site, the design primer has been developed and has been identified the ASM dominant marker of wild-type and two mutants and be used for marker assisted selection.
The present invention realizes through following technical scheme:
A kind of and Chinese cabbage eIF4E.a wild-type and two mutants are identified directly related locus specificity dominance ASM mark: detect relevant mark called after ASM-4E.a-s with the wild-type site, clip size 384bp is shown in SEQ ID No.1; Relevant mark called after ASM-4e.a-s is detected in corresponding mutational site, and clip size 383bp is shown in the SEQ ID No.3.
A kind of in addition Chinese cabbage eIF4E.a wild-type is identified directly related locus specificity dominance ASM mark with two mutants: detect relevant mark called after ASM-4E.a-lc with the wild-type site, clip size 348bp is shown in SEQ ID No.2; Relevant mark called after ASM-4e.a-lc is detected in corresponding mutational site, and clip size 347bp is shown in the SEQ ID No.4.
A kind ofly be used to differentiate that the primer of above-mentioned two specific specificity dominance molecule markers is:
Forward primer FW:5'-GAAGCTCCTTACGATCCGTCTTCAC-3';
Forward primer Fm:5'-GGAAGCTCCTTACGATCCGTCTTCT-3';
Reverse primer RWm:5'-TGGCACAGATAGGATCCTCCCAC-3'; Like SEQ ID NO.5,6, shown in 7.
The present invention adopt the homologous clone technology from 4 portions of Chinese cabbages anti-/ be cloned into eIF4E.a gene complete sequence respectively the sense TuMV self-mating system material; With the relatively back difference of finding to have many places SNPs of the eIF4E.a of the B.rapa self-mating system R-O-18 that has reported among 4 sequences and the GenBank, show that the BrA.eIF4E.a of being cloned into is new haplotype.Further through the discovery of deriving of coding region prediction and aminoacid sequence, though wherein the eIF4E.a gene ORF of two parts of materials and the ORF of R-O-18 have the difference of several SNPs, coded product and R-O-18's is in full accord; The ORF of other two parts of materials has then lacked a base at the 250th and has caused the ORF phase shift mutation.The eIF4E.a that confirms a base of disappearance in view of the above is a mutator gene.For the ease of on sepharose, detecting the difference of this base; We have designed two forward primers (FW and Fm according to the mutational site; Wherein FW is used to differentiate wild-type, and Fm is used to differentiate mutant), in downstream conserved regions design a general reverse primer RWm.Guaranteeing under the complete prerequisite of material extracting genome DNA that when FW and RWm combination was increased to genomic dna, all bands that can amplify were exactly wild-type, all can not to expand what band be exactly mutant; Otherwise when Fm and RWm combination was increased to genomic dna, all to expand what band be exactly mutant, and every can not to expand what band be exactly wild-type; In addition, can both expand the material that band when two kinds of combinations is heterozygous.So two kinds of combination of primers are used in combination, corroborate each other, the identification efficiency of wild-type, two mutants and heterozygous is reached 100%.
The screening process of said ASM mark is following:
(1) with the anti-TuMV self-mating system of Chinese cabbage He102,8407 and the responsive self-mating system 06-247 of TuMV, Guan291 be material, adopt the CTAB method, extract both genomic dnas.
(2) according to the eIF4E.a sequence of the B.rapa self-mating system R-O-18 that has reported among the GenBank, upstream and downstream designs primer in the coding region.Employing homologous clone technology, the clone has obtained its eIF4E.a and has checked order respectively from four parts of materials.
(3) the eIF4E.a sequence with gained eIF4E.a and R-O-18 compares respectively, has found that many places SNPs and small segment insert and disappearance; Simultaneously to the coding region prediction with translation product of deriving; Base of disappearance has caused phase shift mutation in the coding region from the eIF4E.a of two parts of sensitive material 06-247 and Guan291 in discovery; Only translation produces 103 amino acid whose little peptides; Though and its coding region of eIF4E.a of other two parts of resistant material He102 and 8407 and R-O-18 have the difference of a few SNPs of place, translation product is in full accord.So according to base deletion site two forward primers of design and a general reverse primer; Two forward primers make up with reverse primer respectively; The four parts of materials that increase again, the result has obtained the amplification that bar has and do not have respectively in four parts, and this is the dominant marker.
Said mark can be used as molecule marker and is applied to evaluation of Chinese cabbage germ plasm resource and breeding assist-breeding, selects to contain the germplasm materials or the transformation offspring of eIF4E.a mutation type among 06-247 or the Guan291.Concrete application mode is: the two cover combination of primers of utilizing the ASM mark are carried out the twice PCR amplification to the genomic dna of individuality to be measured; Detect having or not of amplified fragments; If (FW+RWm) combination amplifies the band of 384bp or 348bp, is wild-type then, can not expanding then is mutant; If perhaps (Fm+RWm) combination amplifies the band of 383bp or 347bp then is mutant, can not expanding then is wild-type; If two combinations can both be expanded and band then be heterozygous.
The invention has the beneficial effects as follows: owing to receive the control of a plurality of genes on the Chinese cabbage eIF4E site, the frequency of polygene simultaneous mutation is lower in a material.If can identify two mutants separately in each site respectively, exploitation then can be passed through convergent cross and binding molecule marker assisted selection to the locus specificity molecule marker that each two mutants detects, and a plurality of mutational sites are aggregated in in a material.The present invention utilize the homologous clone scientific discovery eIF4E.a site a two mutants and developed the molecule marker of identifying this site.Utilize this mark accurately to select to backcross transformation offspring's genotype.This mark also can be used for Chinese cabbage germ plasm resource is carried out examination simultaneously, to seek the abundant more two mutants material of genetic background.Its advantage is specific as follows:
1. the mark directly related with the eIF4E.a site mutation that obtain of the present invention, the result is stable, accurate, easy and simple to handle.Being applicable to the screening of different genetic background Chinese cabbage materials, is a ubiquity mark.It can accurately identify the genotype of Chinese cabbage germ plasm resource in this site, has greatly improved the screening efficiency of Chinese cabbage germ plasm resource at eIF4E.a site mutation body.
2. aspect Chinese cabbage eIF4E gene order and mutant research, only Jenner etc. (2010) has reported the sequence of the self-mating system R-O-18 of B.rapa, and other two mutants does not appear in the newspapers.To the specific mark in this mutational site, can identify for germ plasm resource, with the two mutants material of cultivating different genetic backgrounds the assisted Selection instrument is provided through the screening of means such as backcross transformation.Application of the present invention can be simplified the screening means greatly, shorten the transformation time limit, has avoided the blindness of selecting in the conventional breeding method.
Description of drawings
Fig. 1: the amplification (agarose gel electrophoresis) of four parts of Chinese cabbage material eIF4E.a genes, wherein 1,2,3,4 represent 06-247, Guan291, He102 and 8,407 four parts of Chinese cabbage inbred lines materials respectively, M is dna molecular amount standard DL2000plus.
Fig. 2: from 06-247, Guan291, He102,8407 and the eIF4E.a genome sequence comparison (showing discrepant part among the figure) of the R-O-18 that retrieves of GenBank.
Fig. 3: ORFs (ORF) the sequence alignment figure of the eIF4E.a of four parts of materials and R-O-18 (it is 06-247 and Guan291 base deletion part that the different obvious part of differential among the figure, ellipse are irised out part, and all the other are same sense mutation).
Fig. 4: four kinds of deduced amino acid with from the eIF4E.a aminoacid sequence of R-O-18 relatively (can find out among the figure that 06-247 and Guan291 since the 84th amino acids phase shift mutation take place, and premature termination).
Fig. 5: two cover ASM primers are to the amplification of four parts of Chinese cabbage material genomic dnas, and 1,2,3,4 refer to He102 respectively, and 8407,06-247 and Guan291.
Fig. 6: the detected result of two cover ASM primers in different Chinese cabbage resources.
Fig. 7: two overlap ASM primers detected result to the part individual plant in backcross progeny.
Embodiment
Below in conjunction with embodiment the present invention is further described.Experimental technique among the embodiment if no special instructions, is ordinary method.
The clone of eIF4E.a in embodiment 1, the different Chinese cabbage inbred lines material
1.1 Chinese cabbage extracting genome DNA
(1) the Chinese cabbage seedling leaves is put into the mortar of liquid nitrogen precooling, abundant grind into powder in liquid nitrogen;
(2) treat that the liquid nitrogen volatilization is dried, transfer to immediately in the centrifuge tube of 2ml that every 100mg material adds 0.6ml approximately and is preheated to 65 ℃ CTAB extracting solution, after the thawing, thermal agitation mixing sample, 65 ℃ of water-baths are placed and were made lysis in 40-60 minute;
(3) after cracking finishes, take out sample and make it be cooled to room temperature fully.Add isopyknic chloroform (chloroform), put upside down gently and make mixing, room temperature was placed 10 minutes;
(4) room temperature, centrifugal 15 minutes of 12000rpm;
(5) with the careful sucking-off of pipettor upper strata water, add in the centrifuge tube of new 1.5ml, add the Virahol (1:1 volume) of 500 μ l, abundant mixing, precipitation at room temperature 10min;
(6) 4 ℃, the centrifugal 10min of 12000rpm, careful abandoning supernatant;
(7) the DNA deposition is with 75% washing with alcohol of 1ml.4 ℃, the centrifugal 10min collecting precipitation of 8000rpm;
(8) repeat with DNA deposition of 75% washing with alcohol;
(9) remove supernatant, DNA is deposited in and dries about 10-15 minute on the aseptic technique platform, and it is transparent that DNA shows slightly, and adds the Tris.HCl of the 10mM of proper volume (30-50 μ l), and pH8.0 makes resolution of precipitate (can be placed on 4 ℃ of refrigerator dissolvings spends the night);
(10) ultraviolet spectrophotometer and 1% Agrose detected through gel electrophoresis DNA concentration and quality.
1.2eIF4E.a clone and sequential analysis
(1) the eIF4E.a gene complete sequence (GenBank:HM131206) of retrieval B.rapa self-mating system R-O-18 designs forward and reverse primer respectively at upstream of coding region and downstream,
Forward primer: 5'GTTCGGAGAAGAGAAGACGGAG3'
Reverse primer: 5'AAGATTACAGGCTTTTAAGGCC3' is like SEQ ID NO.17, shown in 18.
(2) pcr amplification: in 20 μ l reaction systems, comprise 1 * TransStart FastPfu buffer; The genomic dna of 20ng; 0.4 the forward and reverse primer of μ M; 0.25mM dNTPmix; The TransStart FastPfu archaeal dna polymerase (TransGenAP221) of 1 unit.PCR cycling condition: 95 ℃ of preparatory sex change 5 minutes; Then be 95 ℃ of sex change 30 seconds, annealed 30 seconds for 57.5 ℃ that 72 ℃ were extended 40 seconds, 35-38 circulation, last 72 ℃ were extended 10 minutes.
(3) electrophoresis detection of PCR product:
PCR gets 10 μ lPCR amplified productions and adds 1 μ l, 10 * loading buffer after finishing, and on 1% sepharose, carries out electrophoresis, and electrophoresis finishes back EB dyeing, and gel imaging system is observed and taken pictures automatically.The result sees Fig. 1.
(4) cloning and sequencing of PCR product
After electrophoresis confirms that the purpose band is increased; Get 1 μ l pcr amplification product and add 1 μ l pEasy-Blunt (TransGen CB101) carrier room temperature connection 10 minutes; Transformed into escherichia coli competent cell Trans1-T1 (TransGen:CD501), transformed bacteria was cultivated about 16 hours in 37 ℃ of inversions on the LB solid plate that contains Kan 50 μ g/ml.Bacterium liquid PCR detects back picking positive colony and entrusts Beijing Bo Shang Bioisystech Co., Ltd to carry out the mensuration of dna sequence dna.
The Chinese cabbage eIF4E.a sequential analysis of (5) being cloned
To be BrA.eIF4E.a1 and BrA.eIF4E.a2 from the unnamed gene of TuMV resistant material He102 and 8407, its sequence be shown in SEQ ID NO.8 and SEQ ID NO.9; To be BrA.eIF4e.a1 and BrA.eIF4e.a2 from the unnamed gene of TuMV sensitive material 06-247 and Guan, sequence be shown in SEQ ID NO.10 and SEQ ID NO.11.The homologous gene sequence of R-O-18 significant difference part relatively is as shown in Figure 2 among they and the GenBank.
1.3 ORF prediction and deduced amino acid
(1) retrieval obtains homogenic ORF among the R-O-18 from GenBank.Splign (http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi textpage=online&level=form) instrument with NCBI; Analyze the ORF of BrA.eIF4E.a1, BrA.eIF4E.a2 and BrA.eIF4e.a1, BrA.eIF4e.a2, its sequence is respectively shown in SEQ ID NO.12-15.
(2) its ORF is carried out sequence alignment, the difference part is as shown in Figure 3.Wherein the ORF difference of He102 and 8407 ORF and R-O-18 part mainly is SNPs.Amino acid whose derivation is carried out with DNAMAN software, and the result finds that it all is to agree sudden change.The ORF of 06-247 and Guan291 has place base deletion, irises out part like Fig. 3 ellipse.Amino acid predicts the outcome as shown in Figure 4, and the eIF4E.a of 06-247 and Guan291 only encodes and produces the little peptide (the normal encoding product is 227 amino-acid residues) of 103 amino-acid residues as a result, and its sequence is shown in SEQ ID NO.16.Two other coded product and R-O-18's is identical.
(3) judge that in view of the above the eIF4E.a from 06-247 and Guan291 is a two mutants.
The exploitation of embodiment 2 dominance ASM marks
2.1 design of primers
The careful relatively genome sequence of BrA.eIF4E.a1, BrA.eIF4E.a2, BrA.eIF4e.a1, BrA.eIF4e.a2; The zone that differs greatly is promptly between 150-600bp; Key is the single base deletion that is positioned at the exon district; So be directed against wild-type and two mutants difference, with Primer Premier 5.0 software design locus specificity primers at this place.Detecting relevant forward primer sequence with two mutants is Fm:5'-GGAAGCTCCTTACGATCCGTCTTCT-3'; Detecting relevant forward primer with wild-type is FW:5'-GGAAGCTCCTTACGATCCGTCTTCAC-3', and general reverse primer is RWm:5'-TGGCACAGATAGGATCCTCCCAC-3'.Primer entrusts the big genome company of Beijing China synthetic.
2.2 the acquisition of ASM mark:
(1) utilize (FW+RWm) and (Fm+RWm) amplification of combination of primers in anti-/ sense material respectively: the preparation of PCR reaction solution and amplification condition are of 1.2 (2) bars.
(2) detection of amplification is of 1.2 (3) bars.
(3) amplification is as shown in Figure 5, and (FW+RWm) primer can amplify the band of 384bp and 348bp in wild-type He102 and 8407, in two mutants 06-247 and Guan291, can not expand band; Otherwise (Fm+RWm) be combined in wild-type He102 and 8407 and can not expand band, in two mutants 06-247 and Guan291, expand the band that 383bp and 347bp.This is a dominance ASM mark.The sequence of four kinds of bands is shown in SEQ ID NO.1-4.
(1) extracting genome DNA of different Chinese cabbage resources: 8 parts of the different Chinese cabbage materials of picked at random, these material extracting genome DNA are of 1.1.
(2) pcr amplification: the preparation of PCR reaction solution and amplification condition are of 1.2 (2) bars.
(3) detection of PCR product is of 1.2 (3) bars.Detected result is as shown in Figure 6, and A:FW+RWm makes up amplification; B:Fm+RWm makes up amplification; M molecular weight standard DL2000,1-8 are eight parts of Chinese cabbage materials.As can be seen from the figure, 1-4 is a wild-type, and 5-8 is a two mutants.Two cover primers reach 100% to the evaluation efficient of homozygous wildtype and two mutants.
(4) sequence verification in eIF4E site is of 1.2.Cloning and sequencing is carried out in eIF4E site complete in the differing materials.
(5) sequencing result shows, utilizes (FW+RWm) combination of primers can in wild-type He102 and 8407, amplify band, and sequencing result shows, the wild-type really in this site, and what can not expand band is mutant.Therefore with any one overlap the Chinese cabbage material that combination of primers can both realize being isozygotied in this site accurate evaluation.Two cover combination of primers are used, and feasible evaluation for heterozygous becomes possibility.Therefore two cover marker combination are used, for the wild/sudden change of identifying the eIF4E.a site and the rate of accuracy reached 100% of heterozygosis/homozygosity.
Embodiment 4.ASM mark is to the detection of backcross progeny
4.1 the preparation of backcross progeny: with He102 is maternal, and 06-247 and Guan291 are male parent, and the artificial hybridization of flower bud phase obtains F1, is maternal with F1, is male parent with 06-247 and Guan291 respectively, and same flower bud phase artificial hybridization obtains F1BC1.
4.2 each individual plant DNA extraction of backcross population: 100 strains of picked at random F1BC1 individual plant, nurse young plants in hothouses, get true leaf seedling stage and extract genomic dna according to 1.1 method.
4.3 each individual plant genotype identification of backcross progeny: with each individual plant genomic dna is template, carries out the detection of pcr amplification and PCR product with above-mentioned combination of primers, and amplification condition and detection method are of 1.2.Part individual plant detected result is as shown in Figure 7, and A:FW+RWm makes up amplification; B:Fm+RWm makes up amplification; M molecular weight standard DL2000,1-8 are 8 F1BC1 individual plants selecting at random.M:DNA molecular weight standard D2000.Can find out from A figure, 1,2,4,6,7th, wild-type, 3,5,8th, two mutants type.
Claims (7)
1. Chinese cabbage eIF4E.a mutational site specific molecular marker is characterized in that: detect relevant mark called after ASM-4E.a-s with the wild-type site, clip size 384bp is shown in SEQ ID No.1; Relevant mark called after ASM-4e.a-s is detected in corresponding mutational site, and clip size 383bp is shown in the SEQ ID No.3.
2. Chinese cabbage eIF4E.a mutational site specific molecular marker is characterized in that: detect relevant mark called after ASM-4E.a-lc with the wild-type site, clip size 348bp is shown in SEQ ID No.2; Relevant mark called after ASM-4e.a-lc is detected in corresponding mutational site, and clip size 347bp is shown in the SEQ ID No.4.
3. the primer of claim 1 or 2 described Chinese cabbage eIF4E.a mutational site specific molecular markers, it is characterized in that: primer sequence is following:
Forward primer FW:5'-GAAGCTCCTTACGATCCGTCTTCAC-3';
Forward primer Fm:5'-GGAAGCTCCTTACGATCCGTCTTCT-3';
Reverse primer RWm:5'-TGGCACAGATAGGATCCTCCCAC-3';
Like SEQ ID NO.5,6, shown in 7.
4. claim 1 or the 2 described Chinese cabbage eIF4E.a mutational site specific molecular markers application in the genotype of differentiating Chinese cabbage eIF4E.a gene locus.
5. the application of the described primer of claim 3 in the genotype of differentiating Chinese cabbage eIF4E.a gene locus.
6. according to claim 4 or 5 described application; It is characterized in that: concrete application mode is: utilize (FW+RWm) combination and (Fm+RWm) make up this two covers combination of primers; The genomic dna of individuality to be measured is carried out the twice PCR amplification, detect having or not of amplified fragments, if (FW+RWm) combination amplifies the band of 384bp or 348bp; Be wild-type then, can not expanding then is mutant; If (Fm+RWm) combination amplifies the band of 383bp or 347bp then is mutant, can not expanding then is wild-type; If two combinations can both be expanded and band then be heterozygous.
7. application according to claim 6 is characterized in that: said pcr amplification is specially: in 20 μ l reaction systems, comprise 1 * TransStart FastPfu buffer; The genomic dna of 20ng; 0.4 the forward and reverse primer of μ M; 0.25mMdNTPmix; The TransStart FastPfu archaeal dna polymerase of 1 unit;
PCR cycling condition: 95 ℃ of preparatory sex change 5 minutes; Then be 95 ℃ of sex change 30 seconds, annealed 30 seconds for 57.5 ℃ that 72 ℃ were extended 40 seconds, 35-38 circulation, last 72 ℃ were extended 10 minutes.
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