CN102643805B - Specific primer pair for assisted identification of tomato yellow leaf curl virus resistant plants and application of specific primer pair - Google Patents
Specific primer pair for assisted identification of tomato yellow leaf curl virus resistant plants and application of specific primer pair Download PDFInfo
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Abstract
The invention discloses a specific primer pair for assisted identification of tomato yellow leaf curl virus resistant plants and application of the specific primer pair. The specific primer pair comprises DNA fragments shown in a sequence 1 in a sequence list and DNA fragments shown in a sequence 2 in the sequence list. The specific primer pair has the advantages of high specificity and high sensitivity, and can be used for quickly, accurately and efficiently, in molecular level, detecting whether the plants belong to tomato yellow leaf curl virus resistant cultivars or not and whether the plants carry a tomato yellow leaf curl virus gene Ty3a or not. The specific primer pair can be conveniently applied to identification of tomato hybrid seed purity and tomato hybrid authenticity. The specific primer pair can be conveniently applied to identification and screening of antiviral breeding materials and breeding of novel antiviral cultivars. The technique of the specific primer pair can powerfully and technically support comprehensive prevention and control of tomato yellow leaf curl virus and safe and continuous production of vegetables in China, especially in Beijing.
Description
Technical field
The special primer that the present invention relates to a kind of assistant identification tomato yellow leaf curl virus resistant plant to and use.
Background technology
Tomato (Solanum lycopersicum) is a kind of important worldwide vegetable crop, occupies critical positions in China's facilities vegetable production.Be example with Beijing, tomato, cucumber, paprike and 4 kinds of fructovegetative facilities of eggplant are produced mu, wherein mu surplus the tomato 12.8 ten thousand surplus the areas 250,000.Therefore, the safety in production of tomato is played very important effect for Beijing the "shopping basket' project.
Virus disease is one of the most common and the most general disease of morbidity in the tomato production, and wherein (Tomato yellow leaf curl virus TYLCV) is seriously threatening the safety in production of tomato to tomato yellow leaf curl virus.Tomato yellow leaf curl virus belongs to geminivirus infection section (Geminiviridae) bean golden mosaic virus and belongs to (Begomoviruses), is a kind of twin dna virus, propagates by " super insect "-Bemisia tabaci, has sudden and unmanageable characteristics.Tomato yellow leaf curl virus can infect various plants such as Solanaceae, pulse family, cash crop such as vegetables such as main harm tomato, capsicum, eggplant, wild cabbage and cucumber and tobacco, particularly heavier in tomato harm, symptom (morbidity characterize) performance of plant of being injured is as follows: poor growth, and plant is obviously downgraded; Top vane shrinkage, leaf periphery upsweep, and the blade thickening diminishes, the leaf area yellow; It is few to bear fruit, and fruit is firmly little, and normally annesl is lost commodity; Endangering can not bloom when serious bears fruit, and causes total crop failure.
Tomato yellow leaf curl virus is the harmful organism of new incoming China in recent years.Took place as far back as Israel, and be diffused into numerous countries and regions such as the Middle East, Mediterranean Sea bank, East Asia, South Asia, Africa, Europe, the U.S., Central America, Australia afterwards rapidly in 1964.Imported province, China south in 2002 into.Autumn in 2005 is imported Jiangsu and Zhejiang Provinces one band the same year in the outburst of Bose City Tianyang County, tomato main producing region, Guangxi town's big area.Imported Henan and Shandong in 2007 into.2008 should disease in the whole nation a plurality of provinces and cities developmenting spread.2009 should disease in partial area, Hebei and the great outburst of Shouguang, Shandong, more than 10 ten thousand mu of areas take place, tomato loses more than 80,000 mu.Summer and autumn in 2009, the county takes place successively in Beijing suburb, the big fruit tomato variety of the main pink colour of planting in Beijing area is mostly to this virus sensitivity, this disease spreads all over districts such as Tongzhou, Daxing, Shunyi, Pinggu, Miyun, Yanqing, Changping, Fangshan, Huairou, Fengtai, Chaoyang, Haidian, the morbidity canopy general diseased plant rate 5-10% in chamber, grave illness canopy chamber diseased plant rate about 20%, indivedual canopy chambers sickness rate reaches more than 90%, cause the serious underproduction or total crop failure, and PD is spreading trend rapidly.
From the experience of the harm of countries in the world tomato yellow leaf curl virus and prevention and control, utilize the resistant gene of tomato plants self, cultivating and promoting the novel anti-virus kind is the fundamental way of most economical effective prevention and control tomato yellow leaf curl virus.How to identify rapidly and accurately pointedly and utilize tomato self resistant gene to cultivate the novel anti-virus kind, and verity and the purity of hybrid of quick and precisely identifying tomato novel anti-virus kind easily, this is the active demand of tomato safety in production.
In current research, 6 tomato yellow leaf curl virus resistance related genes from tomato germ plasm resource, have been identified, called after tomato resisting etiolation curve leaf disease virus gene Ty1(claims the Ty1 gene again successively), tomato resisting etiolation curve leaf disease virus gene Ty2(claim the Ty2 gene again), tomato resisting etiolation curve leaf disease virus gene Ty3(claim the Ty3 gene again), tomato resisting etiolation curve leaf disease virus gene Ty3a(claim the Ty3a gene again), tomato resisting etiolation curve leaf disease virus gene Ty4(claims not only the Ty4 gene) and tomato resisting etiolation curve leaf disease virus gene Ty5(but claim the Ty5 gene), above-mentioned 6 genes are positioned at No. 6 karyomit(e) of tomato successively, o.11 karyomit(e), No. 6 karyomit(e), No. 6 karyomit(e), on No. 3 karyomit(e) and No. 4 karyomit(e), Ty1 gene wherein, Ty2 gene and Ty3a gene have been applied in the cultivation of current disease-resistant variety.According to the resistance performance of material, the Ty3a gene has high resistance for this virus at present, but its concrete sequence the unknown.
Summary of the invention
The special primer that the purpose of this invention is to provide a kind of assistant identification tomato yellow leaf curl virus resistant plant to and use.
It is right to the invention provides the special primer that dna fragmentation is formed shown in the sequence 2 of dna fragmentation shown in the sequence 1 of sequence table and sequence table.
Described special primer is to can be used for the assistant identification tomato to the resistance of tomato yellow leaf curl virus.
The present invention also protects a kind of assistant identification tomato to the method for tomato yellow leaf curl virus resistance; comprise the steps: that the genomic dna with tomato to be measured is template; with described special primer to carrying out pcr amplification; be candidate's the disease-resistant tomato of tomato yellow leaf curl virus if having the specific band of 454bp, tomato to be measured in the pcr amplification product, be not candidate's the susceptible tomato of tomato yellow leaf curl virus if do not have the specific band of 454bp, tomato to be measured in the pcr amplification product.
Described tomato to be measured is tomato material M82 or its filial generation, tomato material Gc171 or its filial generation, No. 13, powder of Soviet Union or its filial generation or No. 2, Luo Manna or its filial generation.Described tomato to be measured specifically can be tomato material M82, tomato material Gc171, tomato material M82 and the cross-fertilize seed of tomato material Gc171 or the filial generation of this cross-fertilize seed.
Described special primer is to can be used for screening the disease-resistant tomato of tomato yellow leaf curl virus.
The present invention also protects a kind of method of screening the disease-resistant tomato of tomato yellow leaf curl virus; comprise the steps: that the genomic dna with tomato to be measured is template; with described special primer to carrying out pcr amplification; if have the specific band of 454bp in the pcr amplification product, tomato to be measured is the disease-resistant tomato of candidate's tomato yellow leaf curl virus.
Described tomato to be measured is tomato material M82 or its filial generation, tomato material Gc171 or its filial generation, No. 13, powder of Soviet Union or its filial generation or No. 2, Luo Manna or its filial generation.Described tomato to be measured specifically can be tomato material M82, tomato material Gc171, tomato material M82 and the cross-fertilize seed of tomato material Gc171 or the filial generation of this cross-fertilize seed.
Whether described special primer carries tomato resisting etiolation curve leaf disease virus gene Ty3a to can be used for the assistant identification tomato.
The present invention also protects a kind of assistant identification tomato whether to carry the method for tomato resisting etiolation curve leaf disease virus gene Ty3a; comprise the steps: that the genomic dna with tomato to be measured is template; with described special primer to carrying out pcr amplification; if have the specific band of 454bp in the pcr amplification product, tomato to be measured is doubtful carries tomato resisting etiolation curve leaf disease virus gene Ty3a, if do not have the specific band of 454bp, the doubtful tomato resisting etiolation curve leaf disease virus gene Ty3a that do not carry of tomato to be measured in the pcr amplification product.
Described tomato to be measured is tomato material M82 or its filial generation, tomato material Gc171 or its filial generation, No. 13, powder of Soviet Union or its filial generation or No. 2, Luo Manna or its filial generation.Described tomato to be measured specifically can be tomato material M82, tomato material Gc171, tomato material M82 and the cross-fertilize seed of tomato material Gc171 or the filial generation of this cross-fertilize seed.
Described special primer is to can be used for screening the tomato of carrying tomato resisting etiolation curve leaf disease virus gene Ty3a.
The present invention also protects a kind of method of screening the tomato of tomato resisting etiolation curve leaf disease virus gene Ty3a; comprise the steps: that the genomic dna with tomato to be measured is template; to carrying out pcr amplification, be candidate's the tomato of carrying tomato resisting etiolation curve leaf disease virus gene Ty3a if having the specific band of 454bp, tomato to be measured in the pcr amplification product with described special primer.
Described tomato to be measured is tomato material M82 or its filial generation, tomato material Gc171 or its filial generation, No. 13, powder of Soviet Union or its filial generation or No. 2, Luo Manna or its filial generation.Described tomato to be measured specifically can be tomato material M82, tomato material Gc171, tomato material M82 and the cross-fertilize seed of tomato material Gc171 or the filial generation of this cross-fertilize seed.
Described special primer is to can be used for preparing test kit; The function of described test kit is following (a) and (b), (c), (d), (e), (f) or (g): (a) the assistant identification tomato is to the resistance of tomato yellow leaf curl virus; (b) the disease-resistant tomato of screening tomato yellow leaf curl virus; (c) whether the assistant identification tomato carries tomato resisting etiolation curve leaf disease virus gene Ty3a; (d) screen the tomato of carrying tomato resisting etiolation curve leaf disease virus gene Ty3a; (e) purity of the cross-fertilize seed seed of the disease-resistant tomato of assistant identification tomato yellow leaf curl virus; (f) variety authentication of the cross-fertilize seed of the disease-resistant tomato of assistant identification tomato yellow leaf curl virus; (g) the disease-resistant tomato breeding of tomato yellow leaf curl virus.
Described tomato is tomato material M82 or its filial generation, tomato material Gc171 or its filial generation, No. 13, powder of Soviet Union or its filial generation or No. 2, Luo Manna or its filial generation.Described tomato to be measured specifically can be tomato material M82, tomato material Gc171, tomato material M82 and the cross-fertilize seed of tomato material Gc171 or the filial generation of this cross-fertilize seed.
Special primer provided by the invention is right, has good, the highly sensitive advantage of specificity, and whether be tomato resisting etiolation curve leaf disease virus disease-resistant variety, whether carry the Ty3a gene if can detect plant accurately, fast and efficiently from molecular level.Primer of the present invention is identified and the evaluation of seeds of hybridized tomato variety authentication being applied to the seeds of hybridized tomato seed purity easily, can be applied to the evaluation and screening of anti-tomato yellow leaf curl virus breeding material and the cultivation of anti-tomato yellow leaf curl virus new variety easily.The present invention will provide strong science and technology support for the safe Sustainable Production of vegetables in the comprehensive prevention and control of tomato yellow leaf curl virus and Beijing and the whole nation.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure among the embodiment 2.
Fig. 2 is the symptom performance figure in seedling stage among the embodiment 3.
Fig. 3 is the agarose gel electrophoresis figure among the embodiment 3.
Fig. 4 is the symptom performance figure that becomes strain among the embodiment 3.
Fig. 5 is the agarose gel electrophoresis figure among the embodiment 4.
Fig. 6 is the agarose gel electrophoresis figure among the embodiment 6.
Fig. 7 is the agarose gel electrophoresis figure among the embodiment 7.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.The method of virus inoculation plant is referring to document among the embodiment: the research of the sick resistance authenticate technology of tomato yellow leaf curl virus, Molecular Plant Breeding, 2011,9 (2): 210-217.
Tomato material M82 is available from U.S. tomato genetic resources center (The C.M.Rick Tomato Genetics Resource Center), germplasm is numbered LA3475, be the tomato yellow leaf curl virus susceptible variety, do not contain any tomato yellow leaf curl virus resistance related gene that comprises the Ty3a gene.
Tomato material Gc171 is available from U.S. tomato genetic resources center (The C.M.Rick Tomato Genetics Resource Center), and germ plasm resource is numbered LA1932, is the tomato yellow leaf curl virus disease-resistant variety, contains the Ty3a gene.
Tomato yellow leaf curl virus: the public can be from the Beijing City Agriculture and Forestry Institute, Inst. of Genetics and Development Biology, CAS or Jiangsu Province Agriculture Science Institute obtain; Reference: evaluation and the research of graft inoculation method of area, Shanghai tomato yellow leaf curl virus disease, genomics and applied biology, 2009,28 (1): 115-118.
Carry out a large amount of challenge tests, Molecular Identification and sequence alignment according to enantiopathy tomato germ plasm resource and susceptible tomato germ plasm resource, design a pair of special primer for the identification of disease-resistant tomato and susceptible tomato to following (the right target sequence of primer is the fragment of Ty3a gene):
Ty3a-1F (sequence 1 of sequence table): 5 '-CAATAGTTCTAGAATGGGTAAAA-3 ';
Ty3a-1R (sequence 2 of sequence table): 5 '-GTGTGATATGAGAGGTAGGATT-3 '.
Tomato material M82 and tomato material Gc171 are carried out following experiment respectively:
1, extracts the genomic dna of blade.
2, the genomic dna with step 1 is template, and the primer of forming with Ty3a-1F and Ty3a-1R obtains pcr amplification product to carrying out pcr amplification.
The pcr amplification condition: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30sec, 56 ℃ of annealing 30sec, 72 ℃ are extended 30sec, totally 30 circulations, 72 ℃ are extended 10min.
3, the pcr amplification product with step 2 carries out 1% agarose gel electrophoresis, sees Fig. 1.Among Fig. 1, DL2000 is molecular weight standard, blank is that water replaces genomic dna as the pcr amplification product of template, and Gc171 is the pcr amplification product of template for the genomic dna with tomato material Gc171, and M82 is the pcr amplification product of template for the genomic dna with tomato material M82.
M82 compares with the tomato material, and the pcr amplification product of tomato material Gc171 shows a specific band.
4, the specific band in the recycling step 3 and order-checking, its size is 454bp, shown in the sequence 3 of sequence table.
Above result shows: the special primer of Application Example 1 design is right, according to the specific band that whether has about 454bp in the pcr amplification product, can judge whether tomato to be measured carries the Ty3a gene, if have the specific band of about 454bp, tomato to be measured is carried the Ty3a gene, if do not have the specific band of about 454bp, tomato to be measured is not carried the Ty3a gene, that is to say the right target sequence of the special primer of embodiment 1 design for and the closely linked flag sequence of Ty3a gene.
Tomato material M82 and tomato material Gc171 are carried out following experiment respectively:
One, inoculation tomato yellow leaf curl virus
Inoculate 20 strain tomato seedling with tomato yellow leaf curl virus, as experimental group; Replace tomato yellow leaf curl virus to inoculate 20 strain tomato seedling with the equal-volume sterilized water, in contrast group.
Two, morphologic observation in seedling stage and morbidity statistics
Inoculate after 20 days, observation phenotype in seedling stage is also taken pictures, and the plant that the morbidity sign occurs is disease plant, calculates sickness rate.
In the experimental group, the sickness rate of tomato material M82 is 100%, and the sickness rate of tomato material Gc171 is 0%.In the control group, the sickness rate of tomato material M82 is 0%, and the sickness rate of tomato material Gc171 is 0%.In the experimental group, the photo of tomato material Gc171 is seen Fig. 2 A.In the experimental group, the photo of tomato material M82 is seen Fig. 2 B.
Three, Molecular Identification
Inoculate after 20 days, get three strain plant at random for every group and carry out Molecular Identification, method is with embodiment 2.
1% agarose gel electrophoresis figure sees Fig. 3.Among Fig. 3, DL2000 is molecular weight standard, blank is that water replaces genomic dna as the pcr amplification product of template, negative control is the pcr amplification product of template for the genomic dna with other susceptible tomato, positive control is the pcr amplification product of template for the genomic dna with other disease-resistant tomato, the genomic dna that three Gc171 are respectively with three strain tomato material Gc171 is the pcr amplification product of template, and the genomic dna that three M82 are respectively with tomato material M82 is the pcr amplification product of template.
For tomato material M82, the blade of the blade of experimental group plant and control group plant does not all show the specific band of about 454bp.For tomato material Gc171, the blade of the blade of experimental group plant and control group plant all shows the specific band of about 454bp (sequencing result shows that each specific band size is 454bp).
Above result shows: the special primer of Application Example 1 design is right, according to the specific band that whether has about 454bp in the pcr amplification product, can judge that tomato to be measured is that the disease-resistant tomato of tomato yellow leaf curl virus still is the susceptible tomato of tomato yellow leaf curl virus, be candidate's the disease-resistant tomato of tomato yellow leaf curl virus if having the specific band of about 454bp, tomato to be measured, if do not have the specific band of about 454bp, tomato to be measured is candidate's the susceptible tomato of tomato yellow leaf curl virus.
Four, become the strain morphologic observation
Inoculate after 40 days, observe into the strain phenotype and take pictures, see Fig. 4.The growth conditions of tomato material Gc171 is normal.Gc171 compares with the tomato material, and bearing fruit of tomato material M82 is few, and fruit is firmly little, normally annesl.
Because tomato yellow leaf curl virus causes catastrophic loss to tomato production, applying antiviral kind is the fundamental way that the tomato safety in production takes place, guarantees the prevention and control disease.The verity of antiviral kind and seed purity are the lifelines of kind of industry, and purity how quick and precisely to identify seed is the primary demand of kind of industry operation and tomato safety in production.
Soviet Union's powder is for No. 13 the tomato yellow leaf curl virus disease-resistant variety (carrying tomato resisting etiolation curve leaf disease virus gene Ty3a) that cultivate the Jiangsu Province Agriculture Science Institute.In order to satisfy seed dealer's demand as early as possible, the primer that utilizes Ty3a-1F and Ty3a-1R to form carries out Molecular Detection (method is with embodiment 2) to No. 13 seeds of 200 Soviet Union's powder to stochastic sampling.
The electrophoresis result of part sample is seen Fig. 5.Among Fig. 5, DL2000 is molecular weight standard, and positive control is the disease-resistant parent of No. 13, powder of Soviet Union; Negative control is the susceptible parent of No. 13, powder of Soviet Union, and all the other swimming lanes are the part sample to be tested.In 200 parts of tomato samples, (sequencing result shows to have the specificity bar of about 454bp in 196 parts the pcr amplification product, each specific band size is 454bp), account for the specific band that does not have about 454bp in 98%, 4 part the pcr amplification product of total sample number.This illustrates that this variety seeds purity is 98%, meets corresponding seed quality criteria, can throw in production.
1,1 strain tomato material M82 and 1 strain tomato material Gc171 are hybridized, F1 is for seed for results.
2, be plant with F1 for cultivating seeds, be F1 for plant.
3, F1 is carried out selfing for plant, F2 is for seed for results.
4, be plant with F2 for cultivating seeds, be F2 for plant.
5, with 168 strain F2 for plant seedling inoculating tomato yellow leaf curl virus.
6, inoculation is after 20 days in the step 5, and observation phenotype in seedling stage is also taken pictures, and the plant that the morbidity sign occurs is disease plant, and it is disease plant that 52 strains are arranged in the 168 strain plant, and all the other are disease-resistant plant.
7, in the step 5 inoculation 20 days after, get plant leaf and carry out Molecular Identification, method is with embodiment 2, step 6 is accredited as the specificity bar that has about 454bp in the pcr amplification product of disease-resistant plant, and (sequencing result shows, each specific band size is 454bp), and step 6 is accredited as the specificity bar that does not all have about 454bp in the pcr amplification product of disease plant.
At the phenomenon of fake and forged seed on the present seed market, utilize the special primer of embodiment 1 design right, can detect the verity of seed quickly and easily.
Show the tomato yellow leaf curl virus of No. 2, Luo Manna in No. 2, the tomato variety Luo Manna in garden and farming family village, rice field, Shouguang City town a situation arises and carry out on the-spot investigation respectively being planted in Shouguang City, Shandong Province vegetables expo, typical tomato yellow leaf curl virus symptom does not appear for No. 2 in the Luo Manna that finds the displaying garden, and typical tomato yellow leaf curl virus symptom (sickness rate 31%) appears in No. 2 part plant of the Luo Manna in farming family village.
Respectively No. 2 kinds of Luo Manna in these two places are sampled, utilize the primer of Ty3a-1F and Ty3a-1R composition to carrying out Molecular Detection (method is with embodiment 2).
The part sample the results are shown in Figure 6.Among Fig. 6, DL2000 is molecular weight standard, and blank is water, and negative control is tomato material M82, and positive control is tomato material Gc171.The result shows all have the specificity bar (sequencing result shows that each specific band size is 454bp) of about 454bp in the pcr amplification product of the sample in displaying garden, and all do not have the specificity bar of about 454bp in the pcr amplification product of the sample in farming family village.The Luo Manna in this explanation farming family village is not for No. 2 same tomato variety with the Luo Manna that shows the garden No. 2, and the Luo Manna in farming family village is for No. 2 fake and forged seed.
Utilize the special primer of embodiment 1 design right, can determine whether contain the Ty3a gene in the breeding material of breeder's collection and transformation quickly and easily, whether be the tomato yellow leaf curl virus disease-resistant variety, improve the accuracy of the seed selection of antiviral kind, accelerate breeding process, promote breeding level.
488 parts of random acquisition tomato germ plasm resources utilize the primer of Ty3a-1F and Ty3a-1R composition to carrying out Molecular Detection (method is with embodiment 2).Partial results is seen Fig. 7.Among Fig. 7, DL2000 is molecular weight standard, and negative control is tomato material M82, and positive control is tomato material Gc171.8 parts of specific bands (sequencing result shows that each specific band size is 454bp) with about 454bp in 488 parts of tomato germ plasm resource.
Above 8 parts of germ plasm resources are connected kind of virus and observe phenotype (method is with embodiment 3), these 8 parts of germ plasm resources are disease-resistant variety really, and this has established basic substance for cultivating antiviral kind.
Claims (10)
1. the special primer of the composition of dna fragmentation shown in the sequence 2 of dna fragmentation and sequence table shown in the sequence 1 of sequence table is right.
The described special primer of claim 1 to the assistant identification tomato to the application in the resistance of tomato yellow leaf curl virus.
3. an assistant identification tomato is to the method for tomato yellow leaf curl virus resistance, comprise the steps: that the genomic dna with tomato to be measured is template, with the described special primer of claim 1 to carrying out pcr amplification, be candidate's the disease-resistant tomato of tomato yellow leaf curl virus if having the specific band of 454bp, tomato to be measured in the pcr amplification product, be not candidate's the susceptible tomato of tomato yellow leaf curl virus if do not have the specific band of 454bp, tomato to be measured in the pcr amplification product.
4. the described special primer of claim 1 is to the application in the disease-resistant tomato of screening tomato yellow leaf curl virus.
5. method of screening the disease-resistant tomato of tomato yellow leaf curl virus, comprise the steps: that the genomic dna with tomato to be measured is template, with the described special primer of claim 1 to carrying out pcr amplification, if have the specific band of 454bp in the pcr amplification product, tomato to be measured is the disease-resistant tomato of candidate's tomato yellow leaf curl virus.
6. whether the described special primer of claim 1 is to carrying the application among the tomato resisting etiolation curve leaf disease virus gene Ty3a the assistant identification tomato.
7. method whether the assistant identification tomato carries tomato resisting etiolation curve leaf disease virus gene Ty3a, comprise the steps: that the genomic dna with tomato to be measured is template, with the described special primer of claim 1 to carrying out pcr amplification, if have the specific band of 454bp in the pcr amplification product, tomato to be measured is doubtful carries tomato resisting etiolation curve leaf disease virus gene Ty3a, if do not have the specific band of 454bp, the doubtful tomato resisting etiolation curve leaf disease virus gene Ty3a that do not carry of tomato to be measured in the pcr amplification product.
8. the described special primer of claim 1 is to the application in the tomato of carrying tomato resisting etiolation curve leaf disease virus gene Ty3a in screening.
9. method of screening the tomato of tomato resisting etiolation curve leaf disease virus gene Ty3a, comprise the steps: that the genomic dna with tomato to be measured is template, to carrying out pcr amplification, be candidate's the tomato of carrying tomato resisting etiolation curve leaf disease virus gene Ty3a if having the specific band of 454bp, tomato to be measured in the pcr amplification product with the described special primer of claim 1.
10. the described special primer of claim 1 is to the application in the preparation test kit; The function of described test kit is following (a) and (b), (c), (d), (e), (f) or (g): (a) the assistant identification tomato is to the resistance of tomato yellow leaf curl virus; (b) the disease-resistant tomato of screening tomato yellow leaf curl virus; (c) whether the assistant identification tomato carries tomato resisting etiolation curve leaf disease virus gene Ty3a; (d) screen the tomato of carrying tomato resisting etiolation curve leaf disease virus gene Ty3a; (e) purity of the cross-fertilize seed seed of the disease-resistant tomato of assistant identification tomato yellow leaf curl virus; (f) verity of the cross-fertilize seed kind of the disease-resistant tomato of assistant identification tomato yellow leaf curl virus; (g) the disease-resistant tomato breeding of tomato yellow leaf curl virus.
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| CN102948336A (en) * | 2012-11-12 | 2013-03-06 | 北京市农林科学院 | Method for inoculating tomatoes with tomato yellow leaf curl viruses |
| CN104221846B (en) * | 2014-09-23 | 2016-06-15 | 杭州市农业科学研究院 | Selection-breeding is held concurrently rich in lycopene the method for resisting etiolation curve leaf disease virus Fructus Lycopersici esculenti new lines |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2193804A1 (en) * | 2000-06-23 | 2003-11-01 | Univ Valencia Politecnica | Detection of tomato yellow leaf curl virus (TYLCV) consists of rapid DNA extraction and application of specific indicating oligonucleotides |
| CN102234643A (en) * | 2010-05-04 | 2011-11-09 | 北京市农林科学院 | Specific primer pair for assisting in identifying tomato yellow leaf curl virus and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2193804A1 (en) * | 2000-06-23 | 2003-11-01 | Univ Valencia Politecnica | Detection of tomato yellow leaf curl virus (TYLCV) consists of rapid DNA extraction and application of specific indicating oligonucleotides |
| CN102234643A (en) * | 2010-05-04 | 2011-11-09 | 北京市农林科学院 | Specific primer pair for assisting in identifying tomato yellow leaf curl virus and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Kakade V M et al.Screening for tomato yellow leaf curl virus(TYLCV) resistance plants using caps marker.《International Journal of Plant Science》.2010,第5卷(第1期),357-359. |
| Screening for tomato yellow leaf curl virus(TYLCV) resistance plants using caps marker;Kakade V M et al;《International Journal of Plant Science》;20101231;第5卷(第1期);357-359 * |
| 李常保等.番茄黄化曲叶病毒的快速分子检测.《遗传》.2012,第34卷(第3期),366-370. |
| 番茄黄化曲叶病毒的快速分子检测;李常保等;《遗传》;20120331;第34卷(第3期);366-370 * |
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