CN105176978B - Tomato yellow leaf curl disease-resistant gene ty 5 molecular labeling primer and its application - Google Patents
Tomato yellow leaf curl disease-resistant gene ty 5 molecular labeling primer and its application Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling primers of tomato yellow leaf curl disease-resistant gene ty 5 and its application.The sequence of molecular labeling primer is to include sense primer to be from 5 ' ends to 3 ' ends：TAACAAAGCCCTCAAAGC；The end of anti-sense primer 5 ' is to 3 ' ends：GTCTCCGAAACGTAATCC.Molecular labeling of the present invention is InDel types, and its nucleotides sequence is classified as shown in SEQ ID No.3 or SEQ ID No.4.The present invention uses it for molecular mark by the tomato materials A VTO 1227 containing the genes of ty 5, the InDel molecular labelings of exploitation and the gene linkage；Molecular labeling of the present invention is a kind of InDel marks, it is not necessary to the analysis of polymorphism is can be carried out by digestion, easy to operate, succinct, experimental cost is small.
Description
Technical field
The invention belongs to biological technical field, it is related to molecular marking technique field, and in particular to a kind of tomato yellowing Qu Ye
Sick disease-resistant gene ty-5 molecular labeling primer and its application.
Background technology
Tomato (Solanum lycopersicum) is vegetable crop important in the world, promote agro based economic development,
Huge effect is played in terms of increasing peasant income.It is vulnerable to the influence of multiple diseases in planting process, causes yield to decline, seed selection
Disease-resistant variety is particularly significant.
Tomato yellow leaf curl (Tomato yellow leaf curl disease TYLCD) is the important disease of tomato
Evil, the disease is by the tomato yellow of geminivirus infection section (Geminiviridae) Begomovirus (Begomovirus)
Change curve leaf disease virus (Tomato yellow leaf curl virus TYLCV) to cause, mainly pass through Bemisia tabaci (Bemisia
Tabaci) propagate.The disease is reported in Israel earliest, with going deep into that international trade is come and gone, and TYLCV is gradually to global range
Extension, in the 1990s, the incoming China of the disease, has spread all over all tomato main producing regions of China, to tomato production at present
Cause to seriously endanger, cause huge economic loss.
After TYLCV harm tomatoes, by cultural control, chemical prevention, not only effect is poor, or even also ecological environment can be made
Into destruction, seed selection and plantation disease-resistant variety are most economical, effective, environmentally friendly prevention and controls.Due to TYLCV itself variation, make
The tomato material that Ty-1 and Ty-2 must be comprised only loses resistance in some regions, and in the tomato variety of domestic market
Do not contain ty-5 genes, in order to accelerate ty-5 gene molecule markers assistant breeding (marker-assisted selection,
MAS process), exploitation and the mark of ty-5 close linkages, the material that seed selection contains ty-5 genes have great importance.
At present in terms of ty-5 linked marker researchs, only determine that 1 CAPS marks SlNAC1 is chain, linkage distance is still failed to understand
Really；(referring to Hutton SF, Scott JW, Schuster DJ.Recessive Resistance to Tomato yellow
leaf curl virus from the Tomato Cultivar Tyking Is Located in the Same Region
As Ty-5on Chromosome 4.Hortscience, 2012,47 (3)：324-327), and the type is being examined
, it is necessary to carry out digestion to PCR primer during survey, experimental cost is high, cumbersome.
The content of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of tomato yellow leaf curl disease-resistant gene
Ty-5 molecular labeling primer, for screening the molecular labeling with TYLCV resistant gene ty-5 close linkages.
To solve the above problems, the technical solution adopted in the present invention is as follows：
Tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling primer, the molecular labeling primer includes
Sense primer is held from 5 ' ends to 3 '：TAACAAAGCCCTCAAAGC；
The end of anti-sense primer 5 ' is to 3 ' ends：GTCTCCGAAACGTAATCC.
Another object of the present invention is the provision of a kind of tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling, solution
Certainly in conventional breeding, Resistance Identification workload is big, cycle length, the problem of cost is high；
Tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling is InDel types, is to utilize above-mentioned eggplant yellow
Leaf curl disease-resistant gene ty-5 molecular labeling primer obtaining to divide with ty-5 close linkages codominance DNA by PCR amplifications
Son mark, the nucleotides sequence of the molecular labeling is classified as shown in SEQ ID No.3 or SEQ ID No.4.
Third object of the present invention is to provide a kind of tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling point
Sub- labeling method, overcomes ty-5 marks to need the defect of digestion, applied molecular biology method, with containing ty-5 transgenic tomatos from
Friendship is that AVTO1227 is material, screening and the molecular labeling of TYLCV resistant gene ty-5 close linkages.
Tomato yellow leaf curl disease-resistant gene ty-5 molecule labelling method, comprises the following steps：
1) tomato dna group DNA is extracted；
2) molecular labeling by template of above-mentioned tomato dna group DNA with above-mentioned tomato yellow leaf curl disease-resistant gene ty-5
Primer enters performing PCR amplification, after being separated to the PCR products reacted, identify, develop the color, detects and judges that tomato yellow leaf curl is anti-
Ospc gene ty-5.
Further, the step 2 described in above-mentioned molecule labelling method) in, PCR reaction systems are：10ng/ μ L template DNAs 1
μL；The μ L of 4pmmol/L molecular labeling primers 1.0；10×PCR Buffer 1.0μL；25mmol/L MgCl21.0μL；10mmol/
L dNTPs0.25μL；5U/μL rTaq DNA Polymerase 0.1μL；ddH2O5.65μL；PCR reacts amplification program：
94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C of extensions 30, totally 38 circulations, last 72 DEG C
Extend 7min.
Fourth object of the present invention is that the molecular labeling primer for providing tomato yellow leaf curl disease-resistant gene ty-5 exists
Applied in tomato yellow leaf curl virus disease resistant gene ty-5 detection and in the molecular mark of ty-5 genes.
Tomato yellow leaf curl virus disease resistant gene ty-5 detection methods：By template of tomato dna group DNA with above-mentioned kind
Eggplant tomato yellow leaf curl China disease-resistant gene ty-5 molecular labeling primer enters performing PCR amplification, and carrying out polyacrylamide to amplified production coagulates
Gel electrophoresis, are recorded to band after electrophoresis, are determined whether according to stripe size containing ty-5 genes；Wherein
PCR reaction systems are：Reaction system μ L of totally 10 μ L, 10ng/ μ L template DNAs 1.0；4pmmol/L molecular labelings draw
The μ L of thing 1.0, the μ L of 10 × PCR Buffer 1.0,25mmol/L MgCl21.0μL、10mmo1/L dNTPs0.25μL、5U/μL
rTaq DNA Polymerase 0.1μL、ddH2O5.65μL；
PCR reacts amplification program：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of anneal 30s, 72 DEG C
Extension 30, totally 38 circulations, last 72 DEG C of extensions 7min.
Compared with prior art, the beneficial effects of the present invention are：
1. molecular labeling primer energy TYLCV resistant gene ty-5 close linkages of the present invention, can use TYLCV resistances
Gene ty-5 screening and detection；
2. molecular labeling of the present invention is a kind of InDel marks, it is widely distributed in using one kind in crop gene group,
The mark of design is inserted or lacked according to base sequence, is codominant marker, it is widely distributed on genome, is marked with CAPS
Note is compared, it is not necessary to the analysis of polymorphism is can be carried out by digestion, easy to operate, succinct, experimental cost is small；
3. the present invention is marked by developing with InDel chain ty-5, screening and ty-5 close linkages in segregating population
Mark, to realize TYLCV molecular marks, accelerate to the tomato variety seed selection process containing ty-5 genes；
4. the molecular labeling of the present invention can be combined with other linked markers, the positions of ty-5 on chromosome are determined,
Finely positioning for the gene and hereafter the clone of the gene is laid a good foundation；
5. the molecular labeling of the present invention is applied to the seed selection to ty-5 gene resistant materials, it is possible to decrease chemical in production process
The administration of agricultural chemicals, mitigates the spread and epidemic of disease, saves production cost, ensures the smooth development of tomato production, improves tomato production
The economic benefit of industry.
Brief description of the drawings
The present invention is described in further detail with reference to the accompanying drawings and detailed description；
Fig. 1 InDel mark ty5-17 in parent and extremely resisted, the AFLP system of pole sense individual plant；Wherein M：100bp
Marker；P1：Disease-resistant parent AVTO1227；P2：Susceptible parent moneymaker；1-5：The extremely disease-resistant individual plants of F2；6-10：Feel F2 poles
Sick individual plant；
Fig. 2 InDel mark ty5-17 in parent and the AFLP system of any 37 plants of F2 individual plants；Wherein M：100bp
marker；P1：Disease-resistant parent AVTO1227；P2：Susceptible parent moneymaker；F2 individual plants；By moneymaker ×
Any 37 plants of F2 individual plants that AVTO1227 F1 selfings are obtained；
Fig. 3 InDel mark AFLP systems of the ty5-17 in different tomato materials and breed combination；Wherein, M：100bp
marker；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 powder 11；10：Soviet Union's powder 14；11：
moneymaker×AVT O1227。
Embodiment
Tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling primer, the molecular labeling primer includes
Sense primer is held from 5 ' ends to 3 '：TAACAAAGCCCTCAAAGC(SEQ ID No.1)；
The end of anti-sense primer 5 ' is to 3 ' ends：GTCTCCGAAACGTAATCC(SEQ ID No.2).
Specifically, tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling primer is InDel types, can be with
Ty-5 gene close linkages.
Another object of the present invention is the provision of a kind of tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling, solution
Certainly in conventional breeding, Resistance Identification workload is big, cycle length, the problem of cost is high；
Tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling is InDel types, is to utilize above-mentioned eggplant yellow
Leaf curl disease-resistant gene ty-5 molecular labeling primer obtaining to divide with ty-5 close linkages codominance DNA by PCR amplifications
Son mark, the nucleotides sequence of the molecular labeling is classified as shown in SEQ ID No.3 or SEQ ID No.4.
Third object of the present invention is to provide a kind of tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling point
Sub- labeling method, overcomes ty-5 marks to need the defect of digestion, applied molecular biology method, with containing ty-5 transgenic tomatos from
Friendship is that AVTO1227 is material, screening and the molecular labeling of TYLCV resistant gene ty-5 close linkages.
Tomato yellow leaf curl disease-resistant gene ty-5 molecule labelling method, comprises the following steps：
1) tomato dna group DNA is extracted；
2) molecular labeling by template of above-mentioned tomato dna group DNA with above-mentioned tomato yellow leaf curl disease-resistant gene ty-5
Primer enters performing PCR amplification, after being separated to the PCR products reacted, identify, develop the color, detects and judges that tomato yellow leaf curl is anti-
Ospc gene ty-5.
Further, the step 2 described in above-mentioned molecule labelling method) in, PCR reaction systems are：10ng/ μ L template DNAs 1
μL；The μ L of 4pmmol/L molecular labeling primers 1.0；10×PCR Buffer 1.0μL；25mmol/L MgCl21.0μL；10mmol/
L dNTPs0.25μL；5U/μL rTaq DNA Polymerase0.1μL；ddH2O5.65μL；PCR reacts amplification program：94
DEG C pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30, totally 38 circulations, and last 72 DEG C are prolonged
Stretch 7min.
Fourth object of the present invention is that the molecular labeling primer for providing tomato yellow leaf curl disease-resistant gene ty-5 exists
Applied in tomato yellow leaf curl virus disease resistant gene ty-5 detection and in the molecular mark of ty-5 genes.
Tomato yellow leaf curl virus disease resistant gene ty-5 detection methods：By template of tomato dna group DNA with above-mentioned kind
Eggplant tomato yellow leaf curl China disease-resistant gene ty-5 molecular labeling primer enters performing PCR amplification, and carrying out polyacrylamide to amplified production coagulates
Gel electrophoresis, are recorded to band after electrophoresis, are determined whether according to stripe size containing ty-5 genes；Wherein
PCR reaction systems are：Reaction system μ L of totally 10 μ L, 10ng/ μ L template DNAs 1.0；4pmmol/L molecular labelings draw
The μ L of thing 1.0, the μ L of 10 × PCR Buffer 1.0,25mmol/L MgCl21.0μL、10mmol/L dNTPs0.25μL、5U/μL
rTaq DNA Polymerase 0.1μL、ddH2O5.65μL；
PCR reacts amplification program：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of anneal 30s, 72 DEG C
Extension 30, totally 38 circulations, last 72 DEG C of extensions 7min.
The following is specific embodiment of the present invention, in the following embodiments except providing the raw material of explanation or the source of reagent
Outside, other materials and reagent are existing material and reagent, can be obtained by buying pattern；This hair in the following embodiments
Bright described tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling primer is referred to as ty5-17.
Embodiment 1
Tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling, is InDel types, is to utilize following tomato yellows
Change leaf curl disease-resistant gene ty-5 molecular labeling primer：
Sense primer is held from 5 ' ends to 3 '：TAACAAAGCCCTCAAAGC
The end of anti-sense primer 5 ' is to 3 ' ends：GTCTCCGAAACGTAATCC；
The obtained codominance DNA molecular marker with ty-5 close linkages is expanded by PCR；
PCR reaction systems are：Reaction system μ L of totally 10 μ L, 10ng/ μ L template DNAs 1.0；4pmmol/L molecular labelings draw
The μ L of thing 1.0, the μ L of 10 × PCR Buffer 1.0,25mmol/L MgCl21.0μL、10mmol/L dNTPs0.25μL、5U/μL
rTaq DNA Polymerase 0.1μL、ddH2O5.65μL；
PCR reacts amplification program：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of anneal 30s, 72 DEG C
Extension 30, totally 38 circulations, last 72 DEG C of extensions 7min；
The nucleotides sequence of the molecular labeling is classified as SEQ ID No.3.
Embodiment 2
Tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling, is InDel types, is to utilize above-mentioned tomato yellow
Change leaf curl disease-resistant gene ty-5 molecular labeling primer：
Sense primer is held from 5 ' ends to 3 '：TAACAAAGCCCTCAAAGC
The end of anti-sense primer 5 ' is to 3 ' ends：GTCTCCGAAACGTAATCC；
The obtained codominance DNA molecular marker with ty-5 close linkages is expanded by PCR；
PCR reaction systems are：Reaction system μ L of totally 10 μ L, 10ng/ μ L template DNAs 1.0；4pmmol/L molecular labelings draw
The μ L of thing 1.0, the μ L of 10 × PCR Buffer 1.0,25mmol/L MgCl21.0μL、10mmol/L dNTPs0.25μL、5U/μL
rTaq DNA Polymerase 0.1μL、ddH2O5.65μL；
PCR reacts amplification program：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of anneal 30s, 72 DEG C
Extension 30, totally 38 circulations, last 72 DEG C of extensions 7min；
The nucleotides sequence of the molecular labeling is classified as shown in SEQ ID No.4.
Application Example 1
Different cultivars is identified using tomato yellow leaf curl disease-resistant gene ty-5 of the present invention molecular labeling primer
Tomato whether contain ty-5 genes：
1. material to be tested
Male parent (P1)：(Wang Yinlei, Yang Mali, Zhao Liping, et al. contain anti-tomato yellow leaf curl materials A VTO1227
The introduction of Ty-5 transgenic tomato materials, the agriculture journal of evaluation and utilization southwest, 2014, (05)：2090-2094)；Maternal (P2)：
Feel tomato yellow leaf curl material moneymaker；Colony：Moneym aker × AVTO1227 hybridization prepares F1, and F1 individual plants are certainly
Hand over and obtain F1 generation segregating population；Specific material and breed combination, which are shown in, refers to table 1.
2. authentication step is as follows：
1) Disease Resistance Identification：
Carry the artificial infection of TYLCVD Bemisia tabacis to the seedling of the heart of 4 leaf 1, inoculation sprays insecticide after one week, it
The tomato seedling after inoculation is colonized in greenhouse afterwards；Investigation plant incidence after 4 weeks is colonized, is divided into according to the morbidity order of severity
0-4 grades：0 grade, without any susceptible symptom；1 grade, top limb edge becomes yellow slightly；2 grades, top leaflet somewhat turns yellow and occurred
The curling of very little；3 grades, large-scale blade turns yellow, and curling is serious, and growth increment is small；4 grades, symptom is very serious, and plant is short
Change, stop growing substantially；
2) extraction of genomic DNA：
The DNA for adding 271 μ L after tomato leaf grinding immediately extracts mixed liquor, 271 μ L karyorhexises liquid and 108 μ L dodecanes
65 DEG C of incubator 45min is placed in after base sodium sarcosinate, mixing；600 μ L chloroform-isoamyl alcohol (24: 1) is added, is gently overturn,
Both are well mixed, 10000rpm in centrifuge is put into and centrifuges 6min；Supernatant is drawn, new centrifuge tube is transferred to, 600 μ L are added
Pre- cold isopropanol, gently shakes up, 12000rpm centrifugation 10min, abandons supernatant；200 μ L 70% ethanol is added, is rinsed 2 times, will
Test tube is placed in be dried at room temperature；200 μ LT1/10E solution are added, DNA is dissolved, -20 DEG C of preservations are placed in；
3) InDel molecular labeling primers ty5-17 analysis：
A. the selection of individual plant is felt in extremely anti-pole
By carrying out Bemisia tabaci inoculation to F2 colonies, resistance of the individual plant to tomato yellow leaf curl is identified, according to morbidity
The order of severity is classified；Choose without disease symptom, sick level is recorded as 05 any individual plants as extremely disease-resistant individual plant；Choose and plant
Strain is downgraded, and grows heavily suppressed, and yellowing leaf curling is serious, and sick level is recorded as 45 any individual plants as extremely susceptible list
Strain；
The analysis of B.InDel linked markers
New InDEL sites are chosen near No. 4 chromosome SlNAC1 of tomato to be analyzed, and determine molecular labeling primer
Ty5-17 has linkage relationship with ty-5 genes, is marked using ty5-17 labeled primers, PCR reaction systems in labeling method
For：The μ L of template DNA 10ng/ μ L 1；The sub- μ L of labeled primer ty5-17 1.0 of 4pmmol/L；10×PCR Buffer 1.0μL；
25mmol/L MgCl21.0μL；10mmol/L dN TPs0.25μL；5U/μL rTaq DNA Polymerase 0.1μL；
ddH2O5.65μL；PCR reacts amplification program：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of annealing 30s,
72 DEG C of extension 30s, totally 38 circulations, last 72 DEG C of extensions 7min.PCR primer is on 6% non-denaturing polyacrylamide gel
Separation identification is carried out, then silver staining colour developing is observed on visible drop instrument, records experimental result；
3) qualification result
By carrying out Resistance Identifications to 244 plants of F2 individual plants, by the disease-resistant plant that is designated as of the state of an illness≤2, the state of an illness >=3 are designated as sense
Sick plant, disease-resistant plant amounts to 59 plants, and disease plant amounts to 185 plants, meets 3: 1 segregation ratio (χ2=0.09), ty-5 is
Single recessive inheritance；Molecular labeling primer ty5-17 in disease-resistant parent AVTO1227, Susceptible parent moneymaker and extremely resist and
Expanded between pole sense individual plant, AVTO1227 and all extremely anti-individual plants only amplify the band that size is 172bp；
Moneymaker amplifies 187bp band；4 plants amplify the band that size is 187bp in 5 pole sense individual plants, and 1 plant amplifies
The heterozygosis banding pattern of 172 and 187 sizes, as a result referring to Fig. 1, wherein M：100bp Marker；P1：Disease-resistant parent AVTO1227；P2：
Susceptible parent moneymaker；1-5：The extremely disease-resistant individual plants of F2；6-10：The extremely susceptible individual plants of F2.Two parent's amplified productions are carried out
Sequencing analysis, it was demonstrated that 15bp nucleotide fragments are inserted into moneymaker materials, cause the generation of polymorphism, 15 of insertion
Base is 5 '-ATAAGTACGTATTGG-3 '.
InDel marks ty5-17 is mono- in disease-resistant parent AVTO1227, Susceptible parent moneymaker and any 37 plants of F2
Expanded between strain, be accredited as disease-resistant individual plant and only amplify the band that size is 172bp；It is accredited as susceptible individual plant amplification
Go out size be 172bp band, or 172 and 187 sizes heterozygosis banding pattern.Phenotype and genotype fit like a glove.As a result referring to figure
2, wherein M：100bp marker；P1：Disease-resistant parent AVTO1227；P2：Susceptible parent moneymaker；F2 individual plants；By
Any 37 plants of F2 individual plants that moneymaker × AVTO1227 F1 selfings are obtained.
InDel molecular labeling primers ty5-17 further applies the identification to tomato material and breed combination.Can from Fig. 3
See, wherein, M：100bp marker；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 powder 11；10：
Soviet Union's powder 14；11：moneymaker×AVTO1227.All tomato materials or kind for not containing ty-5 genes, are only expanded
To the band of single 187bp sizes；AVTO1227 and moneymaker F1 cenospecies amplifies the miscellaneous of 172 and 187 sizes
Crossed belt type.Thus illustrate, molecular labeling primer ty5-17 can apply in the molecular mark of ty-5 genes.
Whether the tomato of different cultivars the results are shown in Table 1 containing ty-5 genetic tests.
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 powder 11 | Ty-3/ty-3 |
| Soviet Union's powder 14 | Ty-3/ty-3 |
| moneymaker×AVTO1227 | Ty-5/ty-5 |
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to limit the scope of protection of the invention with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (6)
1. tomato yellow leaf curl disease-resistant gene ty-5 molecular labeling, it is characterised in that the molecular labeling is InDel marking classes
Type, is the molecular labeling primer with tomato yellow leaf curl disease-resistant gene ty-5, and obtain close with ty-5 is expanded by PC R
Chain codominance DNA molecular marker, the nucleotides sequence of the molecular labeling is classified as shown in SEQ ID No.3 or SEQ ID No.4
The upstream primer sequence of wherein molecular labeling primer is：TAACAAAGCCCTCAAAGC, downstream primer sequence is：
GTCTCCGAAACGTAATCC。
2. tomato yellow leaf curl disease-resistant gene ty-5 molecule labelling method, it is characterised in that the molecule labelling method includes
Following steps：
1) tomato dna group DNA is extracted；
2) performing PCR amplification is entered with the molecular labeling primer described in claim 1 by template of above-mentioned tomato dna group DNA, to PCR
The product of reaction separated, identified, develop the color after, detection determines whether tomato yellow leaf curl disease-resistant gene ty-5.
3. molecule labelling method according to claim 2, it is characterised in that step 2) in, PCR reaction systems are：10ng/
The μ L of μ L template DNAs 1；The μ L of 4pmmol/L molecular labeling primers 1.0；10×PCR Buffer 1.0μL；25mmol/L MgCl21.0
μL；10mmol/L dNTPs0.25μL；5U/μl rTaq DNA Polymerase 0.1μL；ddH2O 5.65μL；PCR reacts
Amplification program is：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C extend 30s, totally 38
Circulation, last 72 DEG C of extensions 7min.
4. tomato yellow leaf curl disease-resistant gene ty-5 as claimed in claim 1 molecular labeling primer is in point of ty-5 genes
Application in sub- marker-assisted breeding.
5. tomato yellow leaf curl disease-resistant gene ty-5 as claimed in claim 1 molecular labeling primer is in detection tomato yellowing
Application in leaf curl viral disease resistant gene ty-5 genes.
6. tomato yellow leaf curl virus disease resistant gene ty-5 detection methods, comprise the following steps：Using tomato dna group DNA as mould
Plate and the tomato yellow leaf curl disease-resistant gene ty-5 described in claim 1 molecular labeling primer enter performing PCR amplification, to amplification
Product carries out polyacrylamide gel electrophoresis, and band after electrophoresis is recorded, determined whether according to stripe size containing ty-5
Gene；Wherein
PCR reaction systems are：Reaction system μ L of totally 10 μ L, 10ng/ μ L template DNAs 1.0；4pmmol/L molecular labeling primers 1.0
μL、10×PCR Buffer 1.0μL、25mmol/L MgCl21.0μL、10mmol/L dNTPs0.25μL、5U/μl rTaq
DNA Polymerase 0.1μL、ddH2O5.65μL；
PCR reacts amplification program：94 DEG C of pre-degeneration 5min；Each circulation 94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C of extensions
30s, totally 38 circulations, last 72 DEG C of extensions 7min.
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| 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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| CN102604941A (en) * | 2012-03-20 | 2012-07-25 | 江苏省农业科学院 | Molecular marker method linked with tomato yellow leaf curl disease resistance gene Ty-2 |
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2015
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604941A (en) * | 2012-03-20 | 2012-07-25 | 江苏省农业科学院 | Molecular marker method linked with tomato yellow leaf curl disease resistance gene Ty-2 |
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| Molecular dissection of Tomato leaf curl virus resistance in tomato line TY172 derived from Solanum peruvianum;Ilana Anbinder等;《Theor Appl Genet》;20090520;第119卷;第519-530页 * |
| Recessive Resistance to Tomato yellow leaf curl virus from the Tomato Cultivar Tyking Is Located in the Same Region as Ty-5 on Chromosome 4;Samuel F. Hutton等;《Hort Science》;20120331;第47卷(第3期);第324-327页 * |
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| 番茄抗黄化曲叶病基因与基因工程研究最新进展;杨晓慧等;《园艺学报》;20121125;第39卷(第11期);第2283-2290页 * |
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Inventor after: Wang Yinlei Inventor after: Zhao Tongmin Inventor after: Zhao Liping Inventor before: Wang Yinlei |