CN105176978A - Molecular marker primer of disease-resistant gene ty-5 of tomato yellow leaf curl disease and application of molecular marker primer - Google Patents
Molecular marker primer of disease-resistant gene ty-5 of tomato yellow leaf curl disease and application of molecular marker primer Download PDFInfo
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Abstract
The invention discloses a molecular marker primer of a disease-resistant gene ty-5 of the tomato yellow leaf curl disease and application of the molecular marker primer. Sequences of the molecular marker primer are TAACAAAGCCCTCAAAGC from 5' terminal to 3' terminal of a forward primer and GTCTCCGAAACGTAATCC from 5' terminal to 3' terminal of a reverse primer. A molecular marker is of an InDel marker type, a nucleotide sequence of the molecular marker is SEQ ID No.3 or SEQ ID No.4. The InDel molecular marker linked with the gene is developed by a tomato material AVTO 1227 containing the gene ty-5 and is used for molecular marker-assisted selection. The molecular marker which is the InDel marker can be used for pleomorphic analysis without being subjected to enzyme digestion, and accordingly, convenience and simplicity in operation and low experiment cost are achieved.
Description
Technical field
The invention belongs to biological technical field, relate to molecular marking technique field, be specifically related to a kind of tomato yellow leaf curl disease-resistant gene ty-5 molecule marker primer and application thereof.
Background technology
Tomato (Solanumlycopersicum) is vegetable crop important in the world, in promotion agro based economic development, increasing peasant income, play huge effect.Be vulnerable to the impact of multiple diseases in planting process, cause production declining, breeding resistant variety is very important.
Tomato yellow leaf curl (TomatoyellowleafcurldiseaseTYLCD) is the important disease of tomato, this disease is caused by the tomato yellow leaf curl virus (TomatoyellowleafcurlvirusTYLCV) of geminivirus infection section (Geminiviridae) Begomovirus (Begomovirus), propagates mainly through Bemisia tabaci (Bemisiatabaci).This disease is reported in Israel the earliest, and along with going deep into of international trade contact, TYLCV expands to global range gradually, the nineties in 20th century, this disease imports China into, has spread all over the tomato main producing region that China is all at present, serious harm is caused to tomato production, causes huge financial loss.
TYLCV by cultural control, chemical prevention not only weak effect, even also can damage ecotope after endangering tomato, and seed selection and plantation disease-resistant variety are prevention and controls that is most economical, effective, environmental protection.Due to the variation of TYLCV self, the tomato material only containing Ty-1 and Ty-2 is made to lose resistance in some regions, and all not containing ty-5 gene in the tomato variety of domestic market, in order to accelerate ty-5 gene molecule marker assistant breeding (marker-assistedselection, MAS) process, exploitation and the closely linked mark of ty-5, the material that seed selection contains ty-5 gene has great importance.
At present in the research of ty-5 linked marker, only determine that 1 CAPS marks SlNAC1 chain, linkage distance is still not clear; (see HuttonSF, ScottJW, SchusterDJ.RecessiveResistancetoTomatoyellowleafcurlviru sfromtheTomatoCultivarTykingIsLocatedintheSameRegionasTy-5onChromosome4.Hortscience, 2012,47 (3): 324-327), and this labeling pattern is when detecting, and needs to carry out enzyme to PCR primer and cuts, experimental cost is high, complex operation.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide the molecule marker primer of a kind of tomato yellow leaf curl disease-resistant gene ty-5, for screening and the closely linked molecule marker of TYLCV resistant gene ty-5.
For solving the problem, the technical solution adopted in the present invention is as follows:
The molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5, this molecule marker primer comprises
Upstream primer from 5 ' end to 3 ' end is: TAACAAAGCCCTCAAAGC;
Downstream primer 5 ' holds 3 ' to hold: GTCTCCGAAACGTAATCC.
Another object of the present invention there are provided the molecule marker of a kind of tomato yellow leaf curl disease-resistant gene ty-5, solves in conventional breeding, the problem that Resistance Identification workload is large, the cycle is long, cost is high;
The molecule marker of tomato yellow leaf curl disease-resistant gene ty-5 is InDel labeling pattern, it is the molecule marker primer utilizing above-mentioned eggplant tomato yellow leaf curl China disease-resistant gene ty-5, through the codominance DAN molecule marker closely linked with ty-5 that pcr amplification obtains, the nucleotides sequence of this molecule marker is classified as shown in SEQIDNo.3 or SEQIDNo.4.
3rd object of the present invention is the molecule marker molecule marking method providing a kind of tomato yellow leaf curl disease-resistant gene ty-5, overcome ty-5 and mark the defect needing enzyme to cut, applied molecular biology method, with containing ty-5 transgenic tomato self-mating system AVTO1227 for material, screening with the closely linked molecule marker of TYLCV resistant gene ty-5.
The molecule marking method of tomato yellow leaf curl disease-resistant gene ty-5, comprises the following steps:
1) tomato dna group DNA is extracted;
2) with above-mentioned tomato dna group DNA for the molecule marker primer of template and above-mentioned tomato yellow leaf curl disease-resistant gene ty-5 carries out pcr amplification, after the product of PCR reaction is separated, identifies, develop the color, detect and judge tomato yellow leaf curl disease-resistant gene ty-5.
Further, the step 2 described in above-mentioned molecule marking method) in, PCR reaction system is: 10ng/ μ L template DNA 1 μ L; 4pmmol/L molecule marker primer 1.0 μ L; 10 × PCRBuffer1.0 μ L; 25mmol/LMgCl
21.0 μ L; 10mmol/LdNTPs0.25 μ L; 5U/ μ LrTaqDNAPolymerase0.1 μ L; ddH
2o5.65 μ L; PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C extend 7min.
4th object of the present invention is provide the molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5 in the detection of the sick resistant gene ty-5 of tomato yellow leaf curl virus and apply in the molecular mark of ty-5 gene.
Tomato yellow leaf curl virus sick resistant gene ty-5 detection method: with tomato dna group DNA for the molecule marker primer of template and above-mentioned tomato yellow leaf curl disease-resistant gene ty-5 carries out pcr amplification, polyacrylamide gel electrophoresis is carried out to amplified production, record is carried out to band after electrophoresis, determines whether containing ty-5 gene according to stripe size; Wherein
PCR reaction system is: reaction system is totally 10 μ L, 10ng/ μ L template DNA 1.0 μ L; 4pmmol/L molecule marker primer 1.0 μ L, 10 × PCRBuffer1.0 μ L, 25mmol/LMgCl
21.0 μ L, 10mmol/LdNTPs0.25 μ L, 5U/ μ LrTaqDNAPolymerase0.1 μ L, ddH
2o5.65 μ L;
PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C extend 7min.
Compared to existing technology, beneficial effect of the present invention is:
1. molecule marker primer energy TYLCV resistant gene ty-5 close linkage of the present invention, can use screening and the detection of TYLCV resistant gene ty-5;
2. molecule marker of the present invention is that a kind of InDel marks, one is utilized extensively to be distributed in crop gene group, according to the mark that base sequence inserts or disappearance designs, for codominant marker, its widely distributed on genome, compared with marking, does not need to cut through enzyme the analysis just can carrying out polymorphism with CAPS, easy to operate, succinct, experimental cost is little;
3. the present invention is marked by the InDel that exploitation is chain with ty-5, and in segregating population, screening and the closely linked mark of ty-5, to realize TYLCV molecular mark, accelerate the tomato variety seed selection process containing ty-5 gene;
4. molecule marker of the present invention can be combined with other linked marker, determines ty-5 position on chromosome, for this gene Fine Mapping and after this clone of this gene is laid a good foundation;
5. molecule marker of the present invention is applied to the seed selection to ty-5 gene resistant material, can reduce using of chemical pesticide in production process, alleviate the spread and epidemic of disease, save production cost, and ensures carrying out smoothly of tomato production, improves the economic benefit of tomato industry.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail;
Fig. 1 InDel marks ty5-17 at parent and AFLP system that is extremely anti-, pole sense individual plant; Wherein M:100bpMarker; P1: disease-resistant parent AVTO1227; P2: Susceptible parent moneymaker; The extremely disease-resistant individual plant of 1-5:F2; The extremely susceptible individual plant of 6-10:F2;
Fig. 2 InDel marks the AFLP system of ty5-17 at parent and any 37 strain F2 individual plants; Wherein M:100bpmarker; P1: disease-resistant parent AVTO1227; P2: Susceptible parent moneymaker; F2 individual plant; Any 37 strain F2 individual plants obtained by the F1 selfing of moneymaker × AVTO1227;
Fig. 3 InDel marks the AFLP system of ty5-17 in different tomato material and breed combination; Wherein, M:100bpmarker; P1: disease-resistant parent AVTO1227; P2: Susceptible parent moneymaker; 1:1106-7-0; 2:9210; 3:9320; 4:FJ-1; 5:CLN2026D; 6:CLN2777A; 7: Nanjing beautiful jade; 8: Nanjing precious jade 9: Soviet Union's No. 11, powder; 10: Soviet Union's No. 14, powder; 11:moneymaker × AVTO1227.
Embodiment
The molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5, this molecule marker primer comprises
Upstream primer from 5 ' end to 3 ' end is: TAACAAAGCCCTCAAAGC (SEQIDNo.1);
Downstream primer 5 ' holds 3 ' to hold: GTCTCCGAAACGTAATCC (SEQIDNo.2).
Concrete, the molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5 is InDel labeling pattern, can with ty-5 gene close linkage.
Another object of the present invention there are provided the molecule marker of a kind of tomato yellow leaf curl disease-resistant gene ty-5, solves in conventional breeding, the problem that Resistance Identification workload is large, the cycle is long, cost is high;
The molecule marker of tomato yellow leaf curl disease-resistant gene ty-5 is InDel labeling pattern, it is the molecule marker primer utilizing above-mentioned eggplant tomato yellow leaf curl China disease-resistant gene ty-5, through the codominance DAN molecule marker closely linked with ty-5 that pcr amplification obtains, the nucleotides sequence of this molecule marker is classified as shown in SEQIDNo.3 or SEQIDNo.4.
3rd object of the present invention is the molecule marker molecule marking method providing a kind of tomato yellow leaf curl disease-resistant gene ty-5, overcome ty-5 and mark the defect needing enzyme to cut, applied molecular biology method, with containing ty-5 transgenic tomato self-mating system AVTO1227 for material, screening with the closely linked molecule marker of TYLCV resistant gene ty-5.
The molecule marking method of tomato yellow leaf curl disease-resistant gene ty-5, comprises the following steps:
1) tomato dna group DNA is extracted;
2) with above-mentioned tomato dna group DNA for the molecule marker primer of template and above-mentioned tomato yellow leaf curl disease-resistant gene ty-5 carries out pcr amplification, after the product of PCR reaction is separated, identifies, develop the color, detect and judge tomato yellow leaf curl disease-resistant gene ty-5.
Further, the step 2 described in above-mentioned molecule marking method) in, PCR reaction system is: 10ng/ μ L template DNA 1 μ L; 4pmmol/L molecule marker primer 1.0 μ L; 10 × PCRBuffer1.0 μ L; 25mmol/LMgCl
21.0 μ L; 10mmol/LdNTPs0.25 μ L; 5U/ μ LrTaqDNAPolymerase0.1 μ L; ddH
2o5.65 μ L; PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C extend 7min.
4th object of the present invention is provide the molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5 in the detection of the sick resistant gene ty-5 of tomato yellow leaf curl virus and apply in the molecular mark of ty-5 gene.
Tomato yellow leaf curl virus sick resistant gene ty-5 detection method: with tomato dna group DNA for the molecule marker primer of template and above-mentioned tomato yellow leaf curl disease-resistant gene ty-5 carries out pcr amplification, polyacrylamide gel electrophoresis is carried out to amplified production, record is carried out to band after electrophoresis, determines whether containing ty-5 gene according to stripe size; Wherein
PCR reaction system is: reaction system is totally 10 μ L, 10ng/ μ L template DNA 1.0 μ L; 4pmmol/L molecule marker primer 1.0 μ L, 10 × PCRBuffer1.0 μ L, 25mmol/LMgCl
21.0 μ L, 10mmol/LdNTPs0.25 μ L, 5U/ μ LrTaqDNAPolymerase0.1 μ L, ddH
2o5.65 μ L;
PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C extend 7min.
Be below specific embodiment of the present invention, in the following embodiments except the source of the raw material or reagent that provide explanation, other materials and reagent are existing material and reagent, can be obtained by buying pattern; The molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5 of the present invention is referred to as ty5-17 in the following embodiments.
embodiment 1
The molecule marker of tomato yellow leaf curl disease-resistant gene ty-5 is InDel labeling pattern, is the molecule marker primer utilizing following tomato yellow leaf curl disease-resistant gene ty-5:
Upstream primer from 5 ' end to 3 ' end is: TAACAAAGCCCTCAAAGC
Downstream primer 5 ' holds 3 ' to hold: GTCTCCGAAACGTAATCC;
Through the codominance DAN molecule marker closely linked with ty-5 that pcr amplification obtains;
PCR reaction system is: reaction system is totally 10 μ L, 10ng/ μ L template DNA 1.0 μ L; 4pmmol/L molecule marker primer 1.0 μ L, 10 × PCRBuffer1.0 μ L, 25mmol/LMgCl
21.0 μ L, 10mmol/LdNTPs0.25 μ L, 5U/ μ LrTaqDNAPolymerase0.1 μ L, ddH
2o5.65 μ L;
PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C extend 7min;
The nucleotides sequence of this molecule marker is classified as SEQIDNo.3.
embodiment 2
The molecule marker of tomato yellow leaf curl disease-resistant gene ty-5 is InDel labeling pattern, is the molecule marker primer utilizing above-mentioned tomato yellow leaf curl disease-resistant gene ty-5:
Upstream primer from 5 ' end to 3 ' end is: TAACAAAGCCCTCAAAGC
Downstream primer 5 ' holds 3 ' to hold: GTCTCCGAAACGTAATCC;
Through the codominance DAN molecule marker closely linked with ty-5 that pcr amplification obtains;
PCR reaction system is: reaction system is totally 10 μ L, 10ng/ μ L template DNA 1.0 μ L; 4pmmol/L molecule marker primer 1.0 μ L, 10 × PCRBuffer1.0 μ L, 25mmol/LMgCl
21.0 μ L, 10mmol/LdNTPs0.25 μ L, 5U/ μ LrTaqDNAPolymerase0.1 μ L, ddH
2o5.65 μ L;
PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C extend 7min;
The nucleotides sequence of this molecule marker is classified as shown in SEQIDNo.4.
application Example 1
Adopt the tomato of the molecule marker primer qualification different varieties of tomato yellow leaf curl disease-resistant gene ty-5 of the present invention whether containing ty-5 gene:
1. for examination material
Male parent (P1): anti-tomato yellow leaf curl materials A VTO1227 (Wang Yinlei, Yang Mali, Zhao Liping, etal. are containing the introduction of Ty-5 transgenic tomato material, evaluation and utilization. southwestern agriculture journal, 2014, (05): 2090-2094); Maternal (P2): sense tomato yellow leaf curl material moneymaker; Colony: moneymaker × AVTO1227 is hybridized preparation F1, F1 individual plant selfing and is obtained F1 generation segregating population; Concrete material and breed combination are shown in and refer to table 1.
2. authentication step is as follows:
1) Disease Resistance Identification:
The seedling of 4 leaf 1 hearts is carried to the artificial inoculation of TYLCVD Bemisia tabaci, spray sterilant after inoculating one week, afterwards postvaccinal tomato seedling is colonizated in booth; Field planting 4 weeks " Invest, Then Investigate " plant incidences, are divided into 0-4 level according to morbidity severity: 0 grade, without any susceptible symptom; 1 grade, top limb edge flavescence slightly; 2 grades, top leaflet turns yellow a little and occurs very little curling; 3 grades, large-scale blade turns yellow, and seriously curling, growth increment is little; 4 grades, symptom is very serious, and plant is downgraded, and substantially stops growing;
2) extraction of genomic dna:
Add the DNA extraction mixed solution of 271 μ L after tomato leaf grinding immediately, 271 μ L karyorhexis liquid and 108 μ L sarcosyls, mixing is placed on the incubator 45min of 65 DEG C; Add the chloroform-isoamyl alcohol (24: 1) of 600 μ L, overturn gently, make both mix, put into the centrifugal 6min of whizzer 10000rpm; Draw supernatant, proceed to new centrifuge tube, add the pre-cold isopropanol of 600 μ L, shake up gently, the centrifugal 10min of 12000rpm, abandons supernatant liquor; Add 70% ethanol of 200 μ L, rinsing 2 times, dries under test tube being placed in room temperature; Add 200 μ LT1/10E solution, DNA is dissolved, be placed in-20 DEG C of preservations;
3) analysis of InDel molecule marker primer ty5-17:
A. choosing of individual plant is felt in extremely anti-pole
By carrying out Bemisia tabaci inoculation to F2 colony, qualification individual plant is to the resistance of tomato yellow leaf curl, and the severity according to morbidity carries out classification; Choose without disease symptom, sick level is recorded as 5 any individual plants of 0 as extremely disease-resistant individual plant; Choose plant to downgrade, growth is subject to serious suppression, and yellowing leaf is seriously curling, and sick level is recorded as 5 any individual plants of 4 as extremely susceptible individual plant;
The analysis of B.InDel linked marker
Near tomato No. 4 karyomit(e) SlNAC1, choose new InDEL site analyze, determine that molecule marker primer ty5-17 and ty-5 gene have linkage relationship, utilize ty5-17 labeled primer to mark, in marking method, PCR reaction system is: template DNA 10ng/ μ L1 μ L; 4pmmol/L labeled primer ty5-171.0 μ L; 10 × PCRBuffer1.0 μ L; 25mmol/LMgCl
21.0 μ L; 10mmol/LdNTPs0.25 μ L; 5U/ μ LrTaqDNAPolymerase0.1 μ L; ddH
2o5.65 μ L; PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30s, totally 38 circulations, and last 72 DEG C extend 7min.PCR primer carries out isolation identification on the non-denaturing polyacrylamide gel of 6%, and the colour developing of silver dye, then observes, records experimental result on visible drop instrument;
3) qualification result
By carrying out Resistance Identification to 244 strain F2 individual plants, the state of an illness≤2 are designated as disease-resistant plant, the state of an illness>=3 be designated as disease plant, disease-resistant plant amounts to 59 strains, and disease plant amounts to 185 strains, meets the segregation ratio (χ of 3: 1
2=0.09), ty-5 is single recessive inheritance; Molecule marker primer ty5-17 is at disease-resistant parent AVTO1227, Susceptible parent moneymaker and extremely resist and increase between pole sense individual plant, and AVTO1227 and all extremely anti-individual plants all only amplify the band that size is 172bp; Moneymaker amplifies the band of 187bp; In 5 poles sense individual plants, 4 strains amplify the band that size is 187bp, and 1 strain amplifies the heterozygosis banding pattern of 172 and 187 sizes, result see Fig. 1, wherein M:100bpMarker; P1: disease-resistant parent AVTO1227; P2: Susceptible parent moneymaker; The extremely disease-resistant individual plant of 1-5:F2; The extremely susceptible individual plant of 6-10:F2.Carry out sequencing analysis to two parent's amplified productions, confirm that the nucleotide fragments of 15bp is inserted into moneymaker material, cause the generation of polymorphism, 15 bases of insertion are 5 '-ATAAGTACGTATTGG-3 '.
InDel marks ty5-17 and increases between disease-resistant parent AVTO1227, Susceptible parent moneymaker and any 37 strain F2 individual plants, is accredited as disease-resistant individual plant and all only amplifies the band that size is 172bp; Be accredited as susceptible individual plant and amplify the band that size is 172bp, or the heterozygosis banding pattern of 172 and 187 sizes.Phenotype and genotype fit like a glove.Result see Fig. 2, wherein M:100bpmarker; P1: disease-resistant parent AVTO1227; P2: Susceptible parent moneymaker; F2 individual plant; Any 37 strain F2 individual plants obtained by the F1 selfing of moneymaker × AVTO1227.
InDel molecule marker primer ty5-17 is applied to the qualification to tomato material and breed combination further.As seen from Figure 3, wherein, M:100bpmarker; P1: disease-resistant parent AVTO1227; P2: Susceptible parent moneymaker; 1:1106-7-0; 2:9210; 3:9320; 4:FJ-1; 5:CLN2026D; 6:CLN2777A; 7: Nanjing beautiful jade; 8: Nanjing precious jade 9: Soviet Union's No. 11, powder; 10: Soviet Union's No. 14, powder; 11:moneymaker × AVTO1227.All tomato material or kinds not containing ty-5 gene, all only amplification is to the band of single 187bp size; The F1 cross-fertilize seed of AVTO1227 and moneymaker amplifies the heterozygosis banding pattern of 172 and 187 sizes.Illustrate thus, molecule marker primer ty5-17 can be applied in the molecular mark of ty-5 gene.
Whether the tomato of different varieties the results are shown in Table 1 containing ty-5 gene test.
Table 1: tomato material and breed combination
| Title material | Disease-resistant gene type |
| AVTO1227 | ty-5/ty-5 |
| moneymaker | Nothing |
| 1106-7-0 | Ty-3/Ty-3 |
| 9210 | Nothing |
| 9320 | Nothing |
| FJ-1 | Nothing |
| CLN2026D | ty-2/ty-2 |
| CLN2777A | Ty-2/Ty-2 |
| Nanjing beautiful jade | Nothing |
| Nanjing precious jade | Nothing |
| Soviet Union's No. 11, powder | Ty-3/ty-3 |
| Soviet Union's No. 14, powder | Ty-3/ty-3 |
| moneymaker×AVTO1227 | Ty-5/ty-5 |
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (7)
1. the molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5, is characterized in that, this molecule marker primer comprises
Upstream primer from 5 ' end to 3 ' end is: TAACAAAGCCCTCAAAGC;
Downstream primer from 5 ' end to 3 ' end is: GTCTCCGAAACGTAATCC.
2. the molecule marker of tomato yellow leaf curl disease-resistant gene ty-5, it is characterized in that, this molecule marker is InDel labeling pattern, it is the molecule marker primer with tomato yellow leaf curl disease-resistant gene ty-5 according to claim 1, through the codominance DAN molecule marker closely linked with ty-5 that pcr amplification obtains, the nucleotides sequence of this molecule marker is classified as shown in SEQIDNo.3 or SEQIDNo.4.
3. the molecule marking method of tomato yellow leaf curl disease-resistant gene ty-5, is characterized in that, this molecule marking method comprises the following steps:
1) tomato dna group DNA is extracted;
2) with above-mentioned tomato dna group DNA for template and molecule marker primer according to claim 1 carry out pcr amplification, after the product of PCR reaction is separated, identifies, develop the color, detect and judged whether tomato yellow leaf curl disease-resistant gene ty-5.
4. molecule marking method according to claim 3, is characterized in that, step 2) in, PCR reaction system is: 10ng/ μ L template DNA 1 μ L; 4pmmol/L molecule marker primer 1.0 μ L; 10 × PCRBuffer1.0 μ L; 25mmol/LMgCl
21.0 μ L; 10mmol/LdNTPs0.25 μ L; 5U/ μ lrTaqDNAPolymerase0.1 μ L; ddH
2o5.65 μ L; PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30s, totally 38 circulations, and last 72 DEG C extend 7min.
5. the application of molecule marker primer in the molecular mark of ty-5 gene of tomato yellow leaf curl disease-resistant gene ty-5 as claimed in claim 1.
6. the molecule marker primer of tomato yellow leaf curl disease-resistant gene ty-5 as claimed in claim 1 is detecting the application in tomato yellow leaf curl virus sick resistant gene ty-5 gene.
7. tomato yellow leaf curl virus sick resistant gene ty-5 detection method, comprise the following steps: with tomato dna group DNA for the molecule marker primer of template and tomato yellow leaf curl disease-resistant gene ty-5 according to claim 1 carries out pcr amplification, polyacrylamide gel electrophoresis is carried out to amplified production, record is carried out to band after electrophoresis, determines whether containing ty-5 gene according to stripe size; Wherein
PCR reaction system is: reaction system is totally 10 μ L, 10ng/ μ L template DNA 1.0 μ L; 4pmmol/L molecule marker primer 1.0 μ L, 10 × PCRBuffer1.0 μ L, 25mmol/LMgCl
21.0 μ L, 10mmol/LdNTPs0.25 μ L, 5U/ μ lrTaqDNAPolymerase0.1 μ L, ddH
2o5.65 μ L;
PCR reacts amplification program: 94 DEG C of denaturation 5min; Each circulation 94 DEG C of sex change 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30s, totally 38 circulations, and last 72 DEG C extend 7min.
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| CN108330204A (en) * | 2017-01-18 | 2018-07-27 | 中国种子集团有限公司 | Identify molecular labeling and its application of tomato yellow leaf curl virus resistant gene |
| CN109207626A (en) * | 2018-10-31 | 2019-01-15 | 宁夏泰金种业股份有限公司 | A kind of the InDel label and identification method of seeds of hybridized tomato purity |
| CN110684857A (en) * | 2019-09-27 | 2020-01-14 | 桂林市蔬菜研究所 | Functional marker of tomato yellow leaf curl disease resistance gene ty-5 and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755133A (en) * | 2016-04-08 | 2016-07-13 | 山东寿光蔬菜种业集团有限公司 | Tomato neck/root rot molecular marker as well as marking method and application thereof |
| CN108330204A (en) * | 2017-01-18 | 2018-07-27 | 中国种子集团有限公司 | Identify molecular labeling and its application of tomato yellow leaf curl virus resistant gene |
| CN109207626A (en) * | 2018-10-31 | 2019-01-15 | 宁夏泰金种业股份有限公司 | A kind of the InDel label and identification method of seeds of hybridized tomato purity |
| CN110684857A (en) * | 2019-09-27 | 2020-01-14 | 桂林市蔬菜研究所 | Functional marker of tomato yellow leaf curl disease resistance gene ty-5 and application thereof |
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Inventor after: Wang Yinlei Inventor after: Zhao Tongmin Inventor after: Zhao Liping Inventor before: Wang Yinlei |