CN106811536B - The InDel molecular labeling isolated with cucumber ZYMV resistant gene - Google Patents

The InDel molecular labeling isolated with cucumber ZYMV resistant gene Download PDF

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CN106811536B
CN106811536B CN201710157376.7A CN201710157376A CN106811536B CN 106811536 B CN106811536 B CN 106811536B CN 201710157376 A CN201710157376 A CN 201710157376A CN 106811536 B CN106811536 B CN 106811536B
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cucumber
indel
zymv
isolated
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CN106811536A (en
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杨义
何欢乐
潘俊松
蔡润
朱文莹
潘健
杜慧
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Shanghai Jiaotong University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The InDel molecular labeling isolated the present invention relates to one with ZYMV resistant gene, is named as InDel-zym1, and nucleotide sequence fragment shown in the nucleotide sequence fragment shown in SEQ ID NO.1 and SEQ ID NO.2 forms；Wherein nucleotide sequence fragment shown in SEQ ID NO.1 is isolated with resistant gene, and nucleotide sequence fragment shown in SEQ ID NO.2 is isolated with susceptible gene.InDel molecular labeling of the invention has high stability, can the assisting sifting easy, quickly, high-throughput applied to cucumber at seedling stage ZYMV resistance single plant；According to SEQ ID NO.5 and SEQ ID No.6, the difference of resistance candidate dna length caused by being mutated as insertion/deletion can also design molecular labeling of the multipair codominant InDel label as ZYMV resistance screening.InDel of the invention lays a good foundation labeled as the molecular mark of ZYMV resistance, this will greatly speed up the process of cucumber ZYMV resistance molecule breeding.

Description

The InDel molecular labeling isolated with cucumber ZYMV resistant gene
Technical field
The present invention relates to technique for gene engineerings, more particularly, to the InDel molecule isolated with cucumber ZYMV resistant gene Label.
Background technique
Cucumber (Cucumis sativus L.) overgrows for Curcurbitaceae (Cucurbitaceae) annual herb climbs plant Object.The north India area of Himalayas southern foot is originated in, existing more than the 2000 years cultivation histories in China, are secondary of cucumber Source center.Cucumber is as one of one of big important vegetable crop in the world ten and the main cultivation vegetable crop in China, as important Vegetable crop always by the attention of breeder.
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, affect the growth and development of plant, reduce the yield and product of cucumber fruits Matter.Prevention and treatment of the prevention and treatment of ZYMV mainly to its communication media, plant once infect ZYMV, at present still can without effective medicament With administer, therefore, fundamentally to solve these problems, need to cultivate High quality and diseases resistance new breeds of cucumbers, with meet grower and Consumer demand.
Conventional resistance breeding is time-consuming and laborious, needs to undergo the process of mostly generation backcrossing and hybridization；Disease occurs to need special Sick nursery carry out inoculated identification, and it is affected by environment larger；Cucumber ZYMV resistance is controlled by single recessive gene, 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 shortening breeding cycle.The fast development of modern biotechnology is educated to be disease-resistant Kind opens new approach, cultivates disease-resistant variety using biotechnology and has become current hot spot.The premise of molecular breeding is to obtain Obtain the functional gene of correlated traits or the molecular labeling with its close linkage.Using molecular marker analysis system, in genetic group Upper identification resistant heredity rule, positioning and clone's ZYMV resistant gene, study the molecular regulation mechanism of its function and resistance, can be with Theoretical foundation is provided for the molecular mark and Molecular design breeding of cucumber resistant gene.Using close with disease-resistant gene Molecular labeling that is chain or isolating carries out molecular marker assisted selection breeding, multiple disease-resistant genes can be integrated into a product Kind, breeding efficiency is significantly improved, shortens breeding time, and disease-resistant dynamics and persistence is greatly improved, this is for training It brings out and meets the cucumber disease-resistant varieties of grower's demand and be of great significance.
ZYMV possesses longer research history, and 1973, Lisa etc. separated from cucurbita pepo for the first time and describe ZYMV, can Perishability propagation is carried out by aphid, subsequent Provvidenti's et al. studies have shown that the ZYMV resistance of cucumber is hidden by a pair Property gene zym/zym control.2013, Amano etc. was to feel ZYMV cucumber self-mating system ' CS-PMR1 ' and anti-ZYMV cucumber self-mating system ' A192-18 ' is that parent constructs F2Group, by zymA192-18It is positioned in the section of No. six chromosome 50kb of cucumber, wherein including Six candidate genes, between parent to the analysis of candidate gene the result shows that, Vacuolar protein sorting- Associated protein 4-like (VPS4-like) gene (Csa6M152960.1) is most possible candidate gene, There are two nucleotide polymorphisms at the First Exon of the gene between two parents, the gene may with the duplication of virus and It is intercellular mobile related.
The studies above shows that cucumber ZYMV resistance is single recessive gene control, current acquired certain linked markers Genetic distance farther out or be not suitable for large-scale groups screening, be unfavorable for the development of molecular mark.Therefore, it finds ZYMV resistant gene close linkage with cucumber, the molecular labeling that isolates, provides good technology branch for its disease-resistant molecular breeding Support.
Summary of the invention
The object of the invention is in order to provide it is a kind of based on cucumber ZYMV resistance candidate gene exploitation with ZYMV resistance The codominance InDel molecular labeling that gene isolates, this label can be clearly showed that using polyacrylamide gel electrophoresis Difference, it is convenient to be applied to ZYMV resistance molecule assistant breeding.
Discovery is sequenced by the resistance candidate gene to the anti-sense cucumber material of ZYMV in the present invention, and including, subregion discovery is anti- The insertion of a 37bp in sick material, is mutated using this insertion/deletion, and the present invention develops one and cucumber ZYMV resistance base Because of the InDel molecular labeling isolated, so as to the foundation of molecular mark system.Molecular labeling of the invention can letter Just, quickly, with high throughput be applied to breeding practice.
The purpose of the present invention can be achieved through the following technical solutions:
One InDel molecular labeling isolated with cucumber ZYMV resistant gene, is named as InDel-zym1, by SEQ ID The composition of nucleotide sequence fragment shown in nucleotide sequence fragment shown in NO.1 and SEQ ID NO.2；Wherein SEQ ID NO.1 Shown in nucleotide sequence fragment isolated with disease-resistant gene, nucleotide sequence fragment shown in SEQ ID NO.2 and susceptible base Because isolating.
Downstream primer shown in InDel-zym1 upstream primer as shown in SEQ ID NO.3 and SEQ ID NO.4 Amplification obtains.
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 base Because sequencing obtains sequence shown in SEQ ID NO.6 in the susceptible self-mating system of multiple cucumber；There is inserting for a 37bp between the two Enter/deletion mutation, be mutated according to this insertion/deletion, can design what multiple such as InDel-zym1 were isolated with ZYMV resistance Codominance InDel molecular labeling.
Above-mentioned SEQ ID NO.1 is the sequence that InDel-zym1 is expanded in disease-resistant parent, is made of 211 nucleotide；SEQ ID NO.2 is the sequence that InDel-zym1 is expanded in Susceptible parent, is made of 174 nucleotide.By it is multiple it is disease-resistant, susceptible from The Molecular Detection analysis of system is handed over, disease-resistant self-mating system amplifies the band of 211bp size, and susceptible self-mating system amplifies The band of 174bp size.By being returned bulk segregant analysis, this label is isolated with ZYMV resistant gene.Therefore, this codominance InDel be marked with the foundation of the molecular labeling assistant system conducive to ZYMV resistance breeding, can answer easy, quickly, with high throughput For breeding practice.
Above-mentioned upstream primer and downstream primer are synthesized by Shanghai Hua Jin Biotechnology Co., Ltd, and synthetic method is conventional raw Object method.
Traditional resistance breeding is time-consuming and laborious, needs the process by mostly generation backcrossing and hybridization.Disease occurs to need special Sick nursery carry out inoculated identification, and it is affected by environment larger.Cucumber ZYMV resistance is 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 period and the difficulty of ZYMV resistance breeding Degree.
Compared with prior art, the of the invention InDel molecular labeling that isolates is codominance, can distinguish homozygote and miscellaneous Zygote can identify the genotype of plant in cucumber at seedling stage, eliminate the step of per generation is selfed during backcrossing is penetrated into；This InDel slug band difference is big, and genotype can be clearly distinguished with 8% polyacrylamide gel electrophoresis, tracked with it Resistant gene, it is not only accurate but also time saving and energy saving, it can be used for the molecular mark of cucumber ZYMV resistance, accelerate cucumber ZYMV anti- The process of property breeding.
Detailed description of the invention
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 size, and susceptible self-mating system is all the band of 174bp size.
Fig. 2 is agarose gel electrophoresis effect of the molecular labeling InDel-zym1 in backcrossing segregating population.As shown in the figure, SA0422 is Susceptible parent, and TMG-1 is disease-resistant parent, F1Represent first-filial generation；R and S respectively represent backcrossing segregating population in The testing result for 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
One, the exploitation and identification of the InDel label based on cucumber ZYMV resistance candidate gene
1, the verifying of Duo Gekang, 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 base Because sequencing obtains sequence shown in SEQ ID NO.6 in the susceptible self-mating system of multiple cucumber；There is inserting for a 37bp between the two Enter/deletion mutation, be mutated accordingly, the present invention designs forward and reverse draw in the two sides of mutation using 5.0 primer-design software of Primer Object.Upstream primer is shown in SEQ ID NO.3, and downstream primer is shown in SEQ ID NO.4, and the amplified band of resistant material is The amplified band of 211bp, susceptible material are 174bp, can quickly distinguish material therefor by polyacrylamide gel electrophoresis Genotype.According to this principle, the present invention is expanded to obtain and be isolated with cucumber ZYMV resistant gene through upstream primer with downstream primer InDel molecular labeling, be named as InDel-zym1, InDel-zym1 nucleotide sequence fragment as shown in SEQ ID NO.1 It is formed with nucleotide sequence fragment shown in SEQ ID NO.2；Wherein nucleotide sequence fragment shown in SEQ ID NO.1 and anti- Ospc gene isolates, and nucleotide sequence fragment shown in SEQ ID NO.2 is isolated with susceptible gene.
PCR amplification is carried out using the Taq DNA Polymerase of TaKaRa company, PCR reaction system is 10 μ L systems. Amplification program are as follows: 98 DEG C of initial denaturation 5min；98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 20s, 32 recycle；4 DEG C of guarantors It deposits.Product after amplification uses 8% Polyacrylamide Gel Electrophoresis PCR result.
Using this InDel labelling technique, electrophoretic analysis verifying has been carried out to multiple anti-, sense self-mating systems.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.The results show that the segment of 211bp is amplified in disease-resistant self-mating system, and susceptible self-mating system amplifies the segment of 174bp, As a result 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 label can be used to screen different anti-, sense kind.
2, group isolates verifying
In order to prove InDel label InDel-zym1 whether with ZYMV resistant gene there are linkage relationship, the present invention utilizes 130 plants of backcrossing segregating population has carried out the verifying of group's linkage analysis to this label.The results show that in group with disease-resistant parent There is the single plant of consistent banding pattern to show as disease-resistant, the plant for having consistent banding pattern with Susceptible parent is susceptible, the plant of heterozygosis banding pattern For susceptible (referring to fig. 2).This result explanation: this is labeled as codominant marker, can distinguish homozygote and heterozygote；Label with ZYMV resistance isolates.
As shown in Fig. 2, electrophoretic band is steady and audible, Fragment Differential can be shown with 8% polyacrylamide gel electrophoresis. So this InDel label has the advantages that 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 single plant, be the molecule mark of ZYMV resistance Note assistant breeding is laid a good foundation, this will greatly speed up the process of cucumber ZYMV resistance molecule breeding.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention Within protection scope.
<110>Shanghai Communications University
<120>the InDel molecular labeling isolated with cucumber ZYMV resistant gene
<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 gene, is named as InDel-zym1, by SEQ ID The composition of nucleotide sequence fragment shown in nucleotide sequence fragment shown in NO.1 and SEQ ID NO.2；Wherein SEQ ID NO.1 Shown in nucleotide sequence fragment isolated with disease-resistant gene, nucleotide sequence fragment shown in SEQ ID NO.2 and susceptible base Because isolating.
2. the InDel molecular labeling according to claim 1 isolated with cucumber ZYMV resistant gene, which is characterized in that Downstream primer shown in InDel-zym1 upstream primer as shown in SEQ ID NO.3 and SEQ ID NO.4 expands to obtain.
3. the InDel molecular labeling according to claim 1 isolated with cucumber ZYMV resistant gene, which is characterized in that The nucleotide sequence of the disease-resistant gene is as shown in SEQ ID NO.5.
4. the InDel molecular labeling according to claim 1 isolated with cucumber ZYMV resistant gene, which is characterized 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 labeling isolated as described in claim 1 with cucumber ZYMV resistant gene, special Sign is, 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 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 labeling isolated as described in claim 1 with cucumber ZYMV resistant gene, feature exist In the InDel molecular labeling is applied to the assisting sifting of cucumber at seedling stage ZYMV resistance single plant.
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CN107674922B (en) * 2017-09-01 2020-07-10 中国农业科学院蔬菜花卉研究所 InDel marker of cucumber mosaic virus disease resistant gene cmv and application thereof
CN109234438B (en) * 2018-10-31 2021-08-03 上海交通大学 Molecular marker Indel-De3 coseparated with cucumber infinite growth type gene
CN109182588B (en) * 2018-10-31 2021-08-03 上海交通大学 Molecular marker Indel-de2 coseparated with cucumber infinite growth type 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
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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