CN106811536A - The InDel molecular labelings isolated with cucumber ZYMV resistant genes - Google Patents
The InDel molecular labelings isolated with cucumber ZYMV resistant genes Download PDFInfo
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Abstract
The present invention relates to an InDel molecular labeling isolated with ZYMV resistant genes, InDel zym1 are named as, the nucleotide sequence fragment shown in nucleotide sequence fragment and SEQ ID NO.2 shown in SEQ ID NO.1 is constituted；Nucleotide sequence fragment wherein shown in SEQ ID NO.1 is isolated with resistant gene, and the nucleotide sequence fragment shown in SEQ ID NO.2 is isolated with susceptible gene.InDel molecular labelings of the invention have high stability, can be with easy, quick, the high-throughout assisting sifting for being applied to cucumber at seedling stage ZYMV resistance individual plants；According to SEQ ID NO.5 and SEQ ID No.6, the difference of the resistance candidate dna length caused by insertion/deletion mutation can also design molecular labeling of the multipair codominant InDel marks as ZYMV resistance screenings.InDel of the invention lays a good foundation labeled as the molecular mark of ZYMV resistances, and this will greatly speed up the process of cucumber ZYMV resistance molecule breedings.
Description
Technical field
The present invention relates to technique for gene engineering, more particularly, to the InDel molecules isolated with cucumber ZYMV resistant genes
Mark.
Background technology
Cucumber (Cucumis sativus L.) climbs plant for Curcurbitaceae (Cucurbitaceae) annual herb overgrows
Thing.The north India area of Himalayas southern foot is originated in, existing more than the 2000 years cultivation history of China, is secondary of cucumber
Source center.Used as one of big important vegetable crop in the world ten, be also that China is main plants one of vegetable crop to cucumber, and it is used as important
Vegetable crop always enjoy breeding man attention.
In recent years, ZYMV (Zucchini Yellow Mosaic Virus, cucumber Zucchini yellow mosaic virus) is to Huang
Melon harm is more serious, and after Cucumber Infected ZYMV, yellow, floral leaf occurs in blade, and blade surface has strumae, when falling ill serious
Leaf-shrinkage, deformity, plant are downgraded, or even dead, have impact on growing for plant, reduce the yield and product of cucumber fruits
Matter.Preventing and treating of the preventing and treating of ZYMV mainly to its communication media, plant once infects ZYMV, at present still can without effective medicament
With administer, therefore, fundamentally to solve these problems, it is necessary to cultivate High quality and diseases resistance new breeds of cucumbers, with meet grower and
Consumer demand.
Conventional resistance breeding is wasted time and energy, it is necessary to experience the process of many generation backcrossings and hybridization；Disease occurs to need specially
Sick nursery carry out inoculated identification, it is and affected by environment larger；Cucumber ZYMV resistances are by single recessive gene control, back cross breeding
Per generation selfing is needed in journey to confirm the infiltration of recessive resistance；Each of which increases the difficulty for cultivating cucumber disease-resistant variety.Molecule
Breeding can greatly accelerate breeding for disease resistance process, significantly shorten breeding cycle.The fast development of modern biotechnology, is disease-resistant educating
Plant and open new approach, cultivating disease-resistant variety using biotechnology has turned into current focus.The premise of molecular breeding is to obtain
Obtain the functional gene or the molecular labeling with its close linkage of correlated traits.Using molecular marker analysis system, in genetic group
Upper identification resistant heredity rule, positioning and clone's ZYMV resistant genes, study the molecular regulation mechanism of its function and resistance, can be with
For the molecular mark and Molecular design breeding of cucumber resistant gene provide theoretical foundation.Using tight with disease-resistant gene
Molecular labeling that is chain or isolating, carries out molecular marker assisted selection breeding, multiple disease-resistant genes can be incorporated into a product
Kind, breeding efficiency is significantly improved, shorten breeding time, and disease-resistant dynamics and persistence is greatly improved, this is for training
Bring out meet grower's demand cucumber disease-resistant varieties it is significant.
ZYMV possesses research history more long, and 1973, Lisa etc. was separated from cucurbita pepo and described ZYMV first, can
The research for carrying out perishability propagation, subsequent Provvidenti et al. by aphid shows that the ZYMV resistances of cucumber are hidden by a pair
Property gene zym/zym control.2013, Amano etc. was feeling ZYMV cucumber self-mating system ' CS-PMR1 ' and anti-ZYMV cucumber self-mating system
' A192-18 ' is that parent builds F2Colony, by zymA192-18It is positioned in the interval of No. six chromosome 50kb of cucumber, wherein including
Six candidate genes, the analysis result to candidate gene between parent shows, Vacuolar protein sorting-
Associated protein 4-like (VPS4-like) gene (Csa6M152960.1) are most possible candidate gene,
Between two parents at the First Exon of the gene exist two nucleotide polymorphisms, the gene may with virus duplication and
It is intercellular mobile related.
The studies above shows that cucumber ZYMV resistances are single recessive gene control, current acquired some linked markers
Genetic distance farther out or be not suitable for large-scale groups screening, be unfavorable for the development of molecular mark.Therefore, find
With cucumber ZYMV resistant genes close linkage, the molecular labeling for isolating, for its disease-resistant molecular breeding provides good technology branch
Support.
The content of the invention
The purpose of the present invention is just to provide for a kind of developed based on cucumber ZYMV resistances candidate gene and ZYMV resistances
The codominance InDel molecular labelings that gene is isolated, this mark can be clearly showed that using polyacrylamide gel electrophoresis
Difference, is conveniently applied to ZYMV resistance molecule assistant breedings.
The present invention is sequenced by the resistance candidate gene to the anti-sense cucumber materials of ZYMV and found, including, subregion discovery is anti-
An insertion of 37bp in sick material, is mutated using this insertion/deletion, and the present invention develops one with cucumber ZYMV resistance bases
Because of the InDel molecular labelings for isolating, so as to the foundation of molecular mark system.Molecular labeling of the invention can letter
Just, quickly, be applied to breeding practice with high throughput.
The purpose of the present invention can be achieved through the following technical solutions：
One InDel molecular labeling isolated with cucumber ZYMV resistant genes, is named as InDel-zym1, by SEQ ID
Nucleotide sequence fragment composition shown in nucleotide sequence fragment and SEQ ID NO.2 shown in NO.1；Wherein SEQ ID NO.1
Shown nucleotide sequence fragment is isolated with disease-resistant gene, the nucleotide sequence fragment shown in SEQ ID NO.2 and susceptible base
Because isolating.
Sense primers of the InDel-zym1 as shown in SEQ ID NO.3 and the anti-sense primer shown in SEQ ID NO.4
Amplification is obtained.
The nucleotide sequence of the disease-resistant gene is as shown in SEQ ID NO.5.
The nucleotide sequence of the susceptible gene is as shown in SEQ ID NO.6.
It is sequenced in the disease-resistant self-mating system of multiple cucumber as candidate gene and obtains sequence shown in SEQ ID NO.5, by candidate's base
Because sequencing obtains sequence shown in SEQ ID NO.6 in the susceptible self-mating system of multiple cucumber；One 37bp's of presence is slotting between the two
Enter/deletion mutation, be mutated according to this insertion/deletion, can design multiple such as InDel-zym1 and ZYMV resistances are isolated
Codominance InDel molecular labelings.
Above-mentioned SEQ ID NO.1 are the sequence that InDel-zym1 is expanded in disease-resistant parent, are made up of 211 nucleotides；SEQ
ID NO.2 are the sequence that InDel-zym1 is expanded in Susceptible parent, are made up of 174 nucleotides.It is disease-resistant, susceptible certainly by multiple
The Molecular Detection analysis of system is handed over, disease-resistant self-mating system amplifies the band of 211bp sizes, and susceptible self-mating system is amplified
The band of 174bp sizes.By being returned bulk segregant analysis, this mark is isolated with ZYMV resistant genes.Therefore, this codominance
InDel be marked with the foundation of the molecular labeling assistant system beneficial to ZYMV resistance breedings, can it is easy, quick, with high throughput should
For breeding practice.
Above-mentioned sense primer and anti-sense primer are synthesized by Shanghai Hua Jin bio tech ltd, and synthetic method is conventional raw
Thing method.
Traditional resistance breeding is wasted time and energy, it is necessary to pass through the process of many generation backcrossings and hybridization.Disease occurs to need specially
Sick nursery carry out inoculated identification, it is and affected by environment larger.Cucumber ZYMV resistances are controlled by single recessive gene, back cross breeding
Per generation selfing is needed in journey to confirm the infiltration of recessive resistance.These factors both increase cycle and the difficulty of ZYMV resistance breedings
Degree.
Compared with prior art, the InDel molecular labelings that isolate of the invention are for codominance, can distinguish homozygote and miscellaneous
Zygote, cucumber at seedling stage can identification plant genotype, eliminate backcrossing penetrate into during often for selfing the step of；This
InDel slugs band difference is big, genotype can be clearly distinguished with 8% polyacrylamide gel electrophoresis, is tracked with it
Resistant gene, it is not only accurate but also time saving and energy saving, can be used for the molecular mark of cucumber ZYMV resistances, accelerate cucumber ZYMV to resist
The process of property breeding.
Brief description of the drawings
Fig. 1 is molecular labeling InDel-zym1 in different anti-, sense self-mating system polyacrylamide gel electrophoresis effects.It is anti-
Sick self-mating system is all the band of 211bp sizes, and susceptible self-mating system is all the band of 174bp sizes.
Fig. 2 is agarose gel electrophoresis effects of the molecular labeling InDel-zym1 in backcrossing segregating population.Shown in figure,
SA0422 is Susceptible parent, and TMG-1 is disease-resistant parent, F1Represent first-filial generation；R and S represent respectively backcrossing segregating population in
The testing result of the disease-resistant and disease plant that machine is selected；M represents Marker.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment
First, the exploitation and identification of the InDel marks based on cucumber ZYMV resistance candidate genes
1st, Duo Gekang, the checking of sense self-mating system
It is sequenced in the disease-resistant self-mating system of multiple cucumber as candidate gene and obtains sequence shown in SEQ ID NO.5, by candidate's base
Because sequencing obtains sequence shown in SEQ ID NO.6 in the susceptible self-mating system of multiple cucumber；One 37bp's of presence is slotting between the two
Enter/deletion mutation, be mutated accordingly, the present invention designs forward and reverse drawing using the primer-design softwares of Primer 5.0 in the both sides of mutation
Thing.Sense primer is that shown in SEQ ID NO.3, anti-sense primer is that the amplified band of resistant material is shown in SEQ ID NO.4
211bp, the amplified band of susceptible material is 174bp, and material therefor can be quickly distinguished by polyacrylamide gel electrophoresis
Genotype.According to this principle, the present invention obtains being isolated with cucumber ZYMV resistant genes through sense primer with anti-sense primer amplification
InDel molecular labelings, be named as InDel-zym2, nucleotide sequence fragments of the InDel-zym2 as shown in SEQ ID NO.1
Constituted with the nucleotide sequence fragment shown in SEQ ID NO.2；Nucleotide sequence fragment wherein shown in SEQ ID NO.1 with it is anti-
Ospc gene is isolated, and the nucleotide sequence fragment shown in SEQ ID NO.2 is isolated with susceptible gene.
Enter performing PCR using the Taq DNA Polymerase of TaKaRa companies to expand, PCR reaction systems are 10 μ L systems.
Amplification program is：98 DEG C of predegeneration 5min；98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 20s, 32 circulations；4 DEG C of guarantors
Deposit.Product after amplification uses 8% Polyacrylamide Gel Electrophoresis PCR results.
Using this InDel labelling technique, sense self-mating system anti-to multiple has carried out electrophoretic analysis checking.Wherein disease-resistant selfing
System has：SA02, SA06, S94, S1003, WI7230, TMG-1, totally 6 disease resistant cucumber self-mating systems；Susceptible self-mating system has：SA03、
SA04, SA05, SA07, SA08, SA09, Xia Meilun, S52, S05, PI197088, S06, SA0422, totally 12 susceptible cucumber from
Hand over system.Result shown, the fragment of 211bp is amplified in disease-resistant self-mating system, and susceptible self-mating system amplifies the fragment of 174bp,
Result is referring to Fig. 1, in Fig. 1,1：SA02,2：SA03,3：SA04,4：SA05,5：SA06,6：SA07,7：SA08,8：SA09,9：
Xia Meilun, 10：S52,11：S05,12：S94,13：PI197088,14：S1003,15：WI7230,16：S06,17：TMG-1,
18：SA0422.Therefore, this InDel marks can be used to screen different anti-, sense kinds.
2nd, colony isolates checking
In order to prove whether InDel marks InDel-zym1 with ZYMV resistant genes has a linkage relationship, the present invention is utilized
130 plants of backcrossing segregating population has carried out colony's linkage analysis checking to this mark.Result shows, with disease-resistant parent in colony
The individual plant for having consistent banding pattern show as it is disease-resistant, with Susceptible parent have consistent banding pattern plant be susceptible, the plant of heterozygosis banding pattern
It is susceptible (referring to Fig. 2).This result explanation：This is labeled as codominant marker, can distinguish homozygote and heterozygote；Mark with
ZYMV resistances are isolated.
As shown in Fig. 2 electrophoretic band is steady and audible, Fragment Differential can be shown with 8% polyacrylamide gel electrophoresis.
So, this InDel mark have the advantages that it is stable, quick and easy to use, while homozygote and heterozygosis can be clearly distinguished
Son.Molecular labeling of the invention can be applied to the assisting sifting that cucumber at seedling stage ZYMV resists, feels individual plant, be the molecule mark of ZYMV resistances
Note assistant breeding is laid a good foundation, and this will greatly speed up the process of cucumber ZYMV resistance molecule breedings.
The above-mentioned description to embodiment is to be understood that and use invention for ease of those skilled in the art.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel announcement of the invention, does not depart from improvement that scope made and modification all should be of the invention
Within protection domain.
<110>Shanghai Communications University
<120>The InDel molecular labelings isolated with cucumber ZYMV resistant genes
<160> 6
<210> 1
<211> 211
<212> DNA
<213>Cucumber（Cucumis sativus L.）
<400> 1
tgtgttcccc ctttgagctg agattttctt catttcacgt ttcagtttga ttagatcatt 60
agatgtgtag tgattacaat tatattctag tttaattata atggtagtga ttacaattat 120
attctagttt aattataatg gtgcttactt tttaaataaa atcatgtcaa agataattgt 180
ctacctttct ttaggtaaga ggcgaccatg g 211
<210> 2
<211> 174
<212> DNA
<213>Cucumber（Cucumis sativus L.）
<400> 2
tgtgttcccc ctttgagctg agattttctt catttcacgt ttcagtttga ttagatcatt 60
agatgtgtag tgattacaat tatattctag tttaattata atggtgctta ctttttaaat 120
aaaatcatgt caaagataat tgtctacctt tctttaggta agaggcgacc atgg 174
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tgtgttcccc ctttgagctg 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400>4
ccatggtcgc ctcttaccta 20
<210> 5
<211> 4774
<212> DNA
<213>Cucumber（Cucumis sativus L.）
<400> 5
atgtatagca atttcaagga gcaagctatt gaatacgtga agcaggcggt acatgaagat 60
aatgctggca attacgcaaa agcctctccg ctgtatataa acgccttgga gtacttcaaa 120
actcatttga agtatgagaa aaatcccaag atcaaggaag ctattaccca gaaattcacc 180
gagtatttgc gccgggctga ggagatacgt gctgttttgg atgatggcgg tcctggtccg 240
gcttccaatg gagatgcagc tgttgcaact aaacccaaga cgaaacctaa ggatggagaa 300
ggaggagatg gagaggatcc agaacaggct aagttacggg ctggtcttaa ttccgcaatc 360
ataagggaga aaccggatgt taagtggaac gatgttgctg gattggagag cgccaagcag 420
gcgttgcaag aggctgtaat attacccgtt aagttccccc agttctttac tggttagttc 480
cattttgctt ctttcgatct actaccgtat tgatttttgt tgtctcatgt cagtacagtt 540
gtgttccccc tttgagctga gattttcttc atttcacgtt tcagtttgat tagatcatta 600
gatgtgtagt gattacaatt atattctagt ttaattataa tggtagtgat tacaattata 660
ttctagttta attataatgg tgcttacttt ttaaataaaa tcatgtcaaa gataattgtc 720
tacctttctt taggtaagag gcgaccatgg cgagctttct tgttatatgg gcctcctgga 780
actggaaagt catacttggc aaaggctgtt gcgacagagg ctgactcgac atttttcagg 840
taacgatcct ctgaggtttg agtatatgaa cttgtcttgt gtgatttgta tggtagccat 900
atatttctta actttcctag ccattatctt ttaacgtgcc tgtgtgagca acttacaacc 960
cttgagttct catgaaactt tttttatgtc atcaacaaca ttcatgaact ctgtgttgtt 1020
ttctttattc ttatgaaatt gcagcaagta tgaacaatat cattttaaag tggtgtctct 1080
aattaaatat ttttcatgca ttagagttga ggatattagg aattattagc agatatggcc 1140
gtagggaatt tcattttcta aatgttctat ttaaccattt ctcaagattt aatgattcga 1200
agttagatgg cctgttgctc tactgcttta tgggaggtct cctaggcctt cattcatttt 1260
gttggtatag tgtaatttgt tgtcgtcatt gcaattcttt tgttctattt tgttcattta 1320
ttaattcttg aatttggtgg gctttaaatc ttgtttctat agaagaacaa taatggagat 1380
atttgttgat tactcttaag agttttagat ggttagattg gaccatctgt ttgtttggca 1440
ggaaacttaa gaggaacata tgactaaagg taaaggtaag tgctgaagcc ggaagtttaa 1500
tataatttat aaacggtttg gcaaagtagc aattttaatt cagaagatgc atttgtgtca 1560
cattttaagt ggtacaagtg caacctaccc tttctttttc tctggataag caaaagacaa 1620
caaatgctat tgaatctttt cttctacaat ttttatagta aaaaacttat atccttttct 1680
gggtgtggcc tttgagacat tgaaataaga aatccctaga ttcaaattgt gatcttccat 1740
tttttacttg cttcttgaac ttattacggt tcttggcttt gaaaaaaatt tgaggaatac 1800
agtaagctat ctttttaata atttacttta ttgaaataaa gttcaaatgc ctttggtggc 1860
tatggacagt atgaactcat tgagtggcct caagtggata taaagtgcgg aatgcaaaaa 1920
ctcaaatgac ttacaaaata tttccaactt ttggttgtaa aaaaataaac gacagatata 1980
aaaacaagtt ttggttatcc attttctttg ctttcattcc catcctattc aattacaggg 2040
catacccaca ccacattcta tttggatact tttggcggtt gtttgaatgt gtttttaatt 2100
atcagtaagc tctatttgct gtttcctgcc acttgcttta aattgagtat tcttttccat 2160
tttctgttga tagcatttct tcttcggacc ttgtctcgaa atggatgggt gaaagcgaga 2220
agctagtttc aaatcttttt caaatggctc gtgatagcgc gccttctatc atctttattg 2280
atgaaataga ttcgttgtgt ggtcaacgag gtgaaggtaa tgaaagtgaa gcttcaagac 2340
gcattaagac tgaacttctt gtgcaaatgc aggtattgtt tgtttgtagt gtaaaagcgt 2400
gaatattttg tgtaccaaag aaaggttgtt gattattatt attatatatg cgtcctttgc 2460
tcatttcatt gtcttgagaa tgctcatttt cagaaaatat ctcattgctg ctgatctaca 2520
tccaagtgtt tttcagccag ttgaaagttg acttaaatta tatctatagg gttttctgat 2580
agtctaatta cttgtttcta tgattctacc catctatttg ggctttaact gcttaaataa 2640
agctttagaa tatctggctt gtttcatagc ttttcggtat cccaagtttt tcaactttga 2700
tatgtgtatt gggttctcta ccttgttcac attttaaaat ttggtagttg ggcattgggc 2760
attgggcatt gggcattgat ggggacctgg atgggaaacc ctggggctta ggggctgtaa 2820
ctcattggca ttttctcttt gctcccaacc cttgaacatc tgttccacac gttacgctgt 2880
ttgtttttgt gtttgcttgc gtacaatgtc tgtattgaat aatttgtcag atgaattttg 2940
atgcatggtg aaacttgctc aatggaaatg tagcttaatt tctaattgca attaaataat 3000
ctactcatgc agcaaaaagt tattctgtta ttttattttg aagaagattt gtaattcatt 3060
tcatgaatag attagggaaa ggagttgtaa attattcgga cgtttggaga gagatttttt 3120
ttgtttatgt attatgtttt ttcttttccc tcaaacaatc aagtttaatg atagctgttt 3180
gattggattt gttagaagct tggattttga actattttta agaatggtcg tcacaagtgc 3240
gtgattccgt aggaaggaac aactttacct tatttaaaga atagttcatc caagtgtgtt 3300
tttatttaat tttctctcag agaaacagaa aactgattta gcattggttt taaactagat 3360
ctgcgaatat tatctctacc ttgttattaa ttttacttga aagctgaact atatatctcg 3420
tgcatttata gggtgtagga cacaatgatc aaaaggttct tgttcttgct gcaacaaata 3480
ctccctatgc tttggatcag gtgaagtaga atttgtctat ttccatatca tgtaaaggat 3540
tacaaaagtt gaatatctga ctgatacgtg ttttttacag gccattcgtc gacgattcga 3600
caagcggata tacattcctc tacctgattt gaaagccaga cagcacatgt tcaaagttct 3660
gagaattcta ttttattttc tccaataaaa aagcaagttt ttttgttgtt gtcatctcca 3720
tgaactttgc taatattcat agatgtattt aaataggtgc atctggggga tacaccacat 3780
aatttaacag aagcagattt tgaaaactta gcacgcaaga cagatggttt ctcgggttca 3840
gatatttcag tttgtgtaag tgtgtaatat ttgttggatg acgatgaact gaaatctttc 3900
attggtggct tgaatctata tcagatatta gttagaatgt agttcatgga tcccttgaat 3960
ttgcaggtga aggatgtgct ctttgaacct gtacgtaaaa ctcaagatgc tatgttcttc 4020
attatgactc ctgatggtat gtgggtacct tgtggaccaa agcaacaagg agctgtccaa 4080
attagcatgc aagagctagc agccaaagga cttgcctcaa aggtacatca catcttaaca 4140
tcctagatga atgtttagat taaaatttag ttattttcaa ctttcacaaa gtgacaagct 4200
tgcagatatg atatacacag actaatatat tttaacactt cttgcattgt gttcgatttg 4260
atgccacttt gtcgatagct caatggtgat gagtctcatg actgataatt ctatgattga 4320
accacaaaca aaattttgaa actattcatc tttcttctat tggtttattg ggctgttttt 4380
gtatgatttc atattttctt taagaaaaaa aaggcatttt atgcattgtt caaatttaag 4440
tttttataca tgcatatatg ttgaagaaga tctataattc gatgagggaa tgctttatca 4500
taatattttg acaaagtcac tggttgatag gtggaattcc attctttata tcgctaaacg 4560
tttggttatt tgggtgcaga ttcttcctcc accaattaca agaacagatt ttgacaaggt 4620
cttagctaga caaaggccta cggtgagtaa atctgatctg gaaattcatg aaaggtttac 4680
aaaagagttt ggggaggaag gttgaggagt gtgttatagt ttttatcgag tcttatctca 4740
atcatcgctg ttgcatacaa ccaattagag ttaa 4774
<210> 6
<211> 4783
<212> DNA
<213>Cucumber（Cucumis sativus L.）
<400> 6
atgtatagca atttcaagga gcaagctatt gaatacgtga agcaggcggt acatgaagat 60
aatgctggca attacgcaaa agcctttcca ctgtatatga acgccttgga gtacttcaaa 120
actcatttga agtatgagaa aaatcccaag atcaaggaag ctattaccca gaaattcacc 180
gagtatttgc gccgggctga ggagatacgt gctgttttgg atgatggcgg tcctggtccg 240
gcttccaatg gagatgcagc tgttgcaact aaacccaaga cgaaacctaa ggatggagaa 300
ggaggagatg gagaggatcc agaacaggct aagttaaggg ctggtcttaa ttccgcaatc 360
ataagggaga aaccggatgt taagtggaac gatgttgctg gattggagag cgccaagcag 420
gcgttgcaag aggctgtaat attacccgtt aagttccccc agttctttac tggttagttc 480
cattttgctt ctttcgatct actaccgtat tgatttttgt tgtctcatgt cagtacagtt 540
gtgttccccc tttgagctga gattttcttc atttcacgtt tcagtttgat tagatcatta 600
gatgtgtagt gattacaatt atattctagt ttaattataa tggtgcttac tttttaaata 660
aaatcatgtc aaagataatt gtctaccttt ctttaggtaa gaggcgacca tggcgagctt 720
tcttgttata tgggcctcct ggaactggaa agtcatactt ggcaaaggct gttgcgacag 780
aggctgactc gacatttttc aggtaacgat cctctgaggt ttgagtatat gaacttgtct 840
tgtgtgattt gtatggtagc catatatttc ttaactttcc tagctattat cttttaatgt 900
gcctgtgtga gcaacttaca acccttgagt tctcatgaaa ctttttttat gtcatcaaca 960
acattcatga actctgtgtt gctttcttta ttcttatgaa attgcagcaa gtatgaacaa 1020
tatcatttta aagtggtgtc tctaattaaa tatttttcat gcattagagt tgaggatatt 1080
aggaattatt agcagatatg gccgtaggga atttcatttt ctaaatgttc tatttaaccg 1140
tttctcaaga tttaatgatt cgaagttaga tggcctgttg ctctactgct ttatgggagg 1200
tctcctaggc cttcattcat tttgttggta tagtgtaatt tgttgtcgtc attgcaattc 1260
ttttgttcta ttttgttcat ttattaattc ttgaatttgg tgggctttaa atcttgtttc 1320
tatagaagaa caataatgga gatatttgtt gattactctt aagagtttta gatggttaga 1380
ttggaccatc tgtttgtttg gcaggaaact taagaggaac atatgactaa aggtaaaggt 1440
aagtgctgaa gccggaagtt taatataatt tataaacggt ttggcaaagt agcaatttta 1500
attcagaaga tgcatttgtg tcacatttta agtggtacaa gtgcaaccta ccctttcttt 1560
ttctctggat aagcaaaaga caacaaatgc tattgaatct tttcttctac aatttttata 1620
gtaaaaaact tatatccttt tctgggtgtg gcctttgaga cattgaaata agaaatccct 1680
agattcaaat tgtgatcttc cattttttac ttgcttcttg aactttttac ggttcttggc 1740
tttgaaaaaa atttgaggaa tacagtaagc tatcttttta ataatttact ttattgaaat 1800
aaagttcaaa tgcctttggt ggctatggac agtttgaact cattgaatag cctcaagtgg 1860
atataaagtg gggaatgcaa aaactaaaat gacttacaaa atatttccaa cttttggttg 1920
taaaaaaata aacgacagat ataaaaacaa gttttggtta tccattttct ctgctttcat 1980
tcccatccta tttaattaca gggcataccc acaccacatt ctatttggat acttttggcg 2040
gttgtttgaa tgtgttttta attatcaata aactctattt gctgtttcct gccacttgct 2100
ttaaattgag tattcttttc cattttctgt tgatagcatt tcttcttcgg accttgtctc 2160
gaaatggatg ggtgaaagcg agaagctagt ttcaaatctt tttcaaatgg ctcgtgatag 2220
cgcgccttct atcatcttta ttgatgaaat agattcgttg tgtggtcaac gaggtgaagg 2280
taatgaaagt gaagcttcaa gacgcattaa gactgaactt cttgtgcaaa tgcaggtatt 2340
gtttgtttgt agtgtaaaag cgtgaatatt ttgtgtacca aagaaaggtt gttgattatt 2400
attattatat atgcgtcctt tgctcatttc attgtcttga gaatgctcat tttcagaaaa 2460
tatctcattg ctgctgatct acatccaagt gtttttcagc cagttgaaag ttgacttaaa 2520
ttatatctgt agggttttct gatagtctaa ttacttgttt ctatgattct acccatctat 2580
ttgggcttta actgcttaaa taaagcttta gaatatctgg cttgtttcat agcttttcgg 2640
tatcccaagt ttttcaactt tgatatgtgt attgggttct ctaccttgtt cacattttaa 2700
aatttggtag ttgggcattg ggcattgggc attgatgggg acctggatgg gaaaccctgg 2760
ggcttagggg ctgtaactca ttggcatttt ctctttgctc ccaacccttg aacatctgtt 2820
caacatgtta cgctgtttgt ttttgtgttt gcttgcgtac aatgtctgta ttgaataatt 2880
tgtcagatga attttgatgc atggtgaaac ttgctcaatg gaaatgtagc ttaatttcta 2940
attgcaatta aataatctac tcatgcagca aaaagttatt ctgttatttt attttgaaga 3000
agatttgtaa ttcatttcat gaatagatta gggaaaggag ttgtaaatta ttcggacgtt 3060
tggagagaga ttttttttgt ttatgtatta tgttttttct tttccctcaa acaatcaagt 3120
ttaatgatag ctgtttgatt ggatttgtta gaagcttgga ttttgaacta tttttaagaa 3180
tggtcgtcac aagtgcgtga ttccgtagga aggaacaact ttaccttatt taaagaatag 3240
ttcatccacg agaggaaagt gaacaagaaa acaaaagaaa acaaaatcat aaaaatagtt 3300
atctaaatcc aagtgtgttt ttatttaatt ttctctcaga gaaacagaaa actgatttag 3360
cattggtttt aaactagatc tgcgaatatt atctctacct tgttattaat tttacttgaa 3420
agctgaacta tatatctcgt gcatttatag ggtgtaggac acaatgatca aaaggttctt 3480
gttcttgctg caacaaatac tccctatgct ttggatcagg tgaagtagaa attgtctatt 3540
tccatatcat gtaaaggatt acagaagttg aatatctgac tgataagtgt tgtttacagg 3600
ccattcgtcg acgattcgac aagcggatat acattcctct acctgatttg aaagccagac 3660
agcacatgtt caaagttctg agaattctat tttattttct ccaataaaaa agcaagtttt 3720
tttgttgttg tcgtctccat gaactttgct aatattcgta gatgtattta aataggtgca 3780
tctgggggat acaccacata atttaacaga agcagatttt gaaaacttag cacgcaagac 3840
agatggtttc tcgggttcag atatttcagt ttgtgtaagt gtgtaatatt tgttggatga 3900
cgatgaactg aaatctttca ttggtggctt gaatctatat cagatattag ttagaatgta 3960
gttcatggat cccttgaatt tgcaggtgaa ggatgtgctc tttgaacctg tacgtaaaac 4020
tcaagatgct atgttcttca ttatgactcc tgatggtatg tgggtacctt gtggaccaaa 4080
gcaacaagga gctgtccaaa ttagcatgca agagttagca gccaaaggac ttgcctcaaa 4140
ggtacatcac atcttaacat cctagatgaa tgtttagatt aaaatttagt tattttcaac 4200
tttcacaaag tgacaagctt gcagatatga taatatacac agactaatat attttaacac 4260
ttcttgcatt gtgttcgatt tgatgccact ttgtcgatag ctcaatggtg atgagtctca 4320
tgactgataa ttctatgatt gaaccacaaa caaaattttg aaactattca tctttattct 4380
attggtttat tgggccgttt ttgtatgatt tcatattttc tttaagaaaa aaaaggcatt 4440
ttatgcattg ttcaaattta agtttttata catgcatata tgttgaagaa gatctataat 4500
tcgatgaggg aatgctttat aataatattt tgacaaagtc actggttgat aggtggaatt 4560
ccattcttta tatcgctaaa cgtttggtta tttgggtgca gattcttcct ccaccaatta 4620
caagaacaga ttttgacaag gtcttagcta gacaaaggcc tacggtgagt aaatctgatc 4680
tggaaattca tgaaaggttt acaaaagagt ttggggagga aggttgagga gtgtgttata 4740
gtttttatcg agtcttatct caatcatcgc tgttgcatac aac 4783
Claims (6)
1. an InDel molecular labeling isolated with cucumber ZYMV resistant genes, is named as InDel-zym1, by SEQ ID
Nucleotide sequence fragment composition shown in nucleotide sequence fragment and SEQ ID NO.2 shown in NO.1；Wherein SEQ ID NO.1
Shown nucleotide sequence fragment is isolated with disease-resistant gene, the nucleotide sequence fragment shown in SEQ ID NO.2 and susceptible base
Because isolating.
2. the InDel molecular labelings that according to claim 1 and cucumber ZYMV resistant genes are isolated, it is characterised in that
Sense primers of the InDel-zym1 as shown in SEQ ID NO.3 and the anti-sense primer amplification shown in SEQ ID NO.4 are obtained.
3. the InDel molecular labelings that according to claim 1 and cucumber ZYMV resistant genes are isolated, it is characterised in that
The nucleotide sequence of the disease-resistant gene is as shown in SEQ ID NO.5.
4. the InDel molecular labelings that according to claim 1 and cucumber ZYMV resistant genes are isolated, it is characterised in that
The nucleotide sequence of the susceptible gene is as shown in SEQ ID NO.6.
5. the preparation method of the InDel molecular labelings for being isolated with cucumber ZYMV resistant genes as claimed in claim 1, it is special
Levy and be, be sequenced in the disease-resistant self-mating system of multiple cucumber as candidate gene and obtain sequence shown in SEQ ID NO.5, by candidate gene
Sequencing obtains sequence shown in SEQ ID NO.6 in the susceptible self-mating system of multiple cucumber, there is insertion/deletion mutation between the two,
InDel-zym1 is designed according to the mutation of this insertion/deletion.
6. the application of the InDel molecular labelings for being isolated with cucumber ZYMV resistant genes as claimed in claim 1, its feature exists
In described InDel molecular labelings are applied to the assisting sifting of cucumber at seedling stage ZYMV resistance individual plants.
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| CN107674922A (en) * | 2017-09-01 | 2018-02-09 | 中国农业科学院蔬菜花卉研究所 | Cucumber anti cucumber mosaic virus ospc gene cmv InDel marks and its application |
| CN109182588A (en) * | 2018-10-31 | 2019-01-11 | 上海交通大学 | The molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene |
| CN109234439A (en) * | 2018-10-31 | 2019-01-18 | 上海交通大学 | The molecular labeling Indel-de1 isolated with cucumber determinate growth gene |
| CN109234438A (en) * | 2018-10-31 | 2019-01-18 | 上海交通大学 | The molecular labeling Indel-De3 isolated with cucumber one dimensional infinite wall gene |
| CN114381539A (en) * | 2020-10-21 | 2022-04-22 | 北京市农林科学院 | Cucurbita pepo ZYMV virus disease molecular marker and method for identifying resistance of Cucurbita pepo ZYMV virus disease |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674922A (en) * | 2017-09-01 | 2018-02-09 | 中国农业科学院蔬菜花卉研究所 | Cucumber anti cucumber mosaic virus ospc gene cmv InDel marks and its application |
| CN109182588A (en) * | 2018-10-31 | 2019-01-11 | 上海交通大学 | The molecular labeling Indel-de2 isolated with cucumber one dimensional infinite wall gene |
| CN109234439A (en) * | 2018-10-31 | 2019-01-18 | 上海交通大学 | The molecular labeling Indel-de1 isolated with cucumber determinate growth gene |
| CN109234438A (en) * | 2018-10-31 | 2019-01-18 | 上海交通大学 | The molecular labeling Indel-De3 isolated with cucumber one dimensional infinite wall gene |
| CN114381539A (en) * | 2020-10-21 | 2022-04-22 | 北京市农林科学院 | Cucurbita pepo ZYMV virus disease molecular marker and method for identifying resistance of Cucurbita pepo ZYMV virus disease |
| CN114381539B (en) * | 2020-10-21 | 2023-11-24 | 北京市农林科学院 | Molecular marker of zucchini ZYMV virus and method for identifying zucchini ZYMV virus resistance |
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