CN104846109B - Molecular marker with cotton verticillium wilt functional gene and application of molecular marker - Google Patents
Molecular marker with cotton verticillium wilt functional gene and application of molecular marker Download PDFInfo
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Abstract
The invention provides a molecular marker with a cotton verticillium wilt functional gene and an application of the molecular marker, and belongs to the field of molecular genetic breeding of crops. A surface receptor protein gene free of an intact reading frame is obtained through BAC (bacterial artificial chromosome) sequencing; nine gene specificity primers are designed by employing the position, which is different from other known gene sequences with verticillium wilt resistance, of the gene; the nine gene specificity primers are used for amplifying DNA (deoxyribonucleic acid) of a verticillium wilt-resistant and susceptible material; a segment with the length of 1549bp is amplified by only one pair of primers Gbvds1549 in all susceptible materials; a nucleotide sequence is as shown in SEQ ID NO.1, but no amplified product exists in a disease-resistant material; and meanwhile, a method of a functional molecular marker is developed on the basis. Through detection of the functional molecular marker, the verticillium wilt resistance of different cotton varieties or materials can be accurately detected; the molecular marker can be applied to screening of hybrid transforming offspring plants of cotton; the breeding efficiency is improved; the breeding and screening cost is significantly reduced; and the materials with relatively good verticillium wilt resistance can be obtained.
Description
First, technical field
The invention provides a kind of molecular labeling of cotton verticillium wilt functional gene and its application, belong to crops molecule something lost
Pass thremmatology field.The anti-sense material of verticillium wilt can be accurately detected, can be applicable to screen cotton hybrid introgressive line plant, be belonged to
Biological technical field.
2nd, background technology
Cotton verticillium wilt is soil-borne disease, and flower bud, bell can be caused to come off in a large number, and when falling ill serious polished rod is come off into, or even
Death, makes cotton underproduction 20-60%, is the main disease of China's Cotton Production.Up to the present, there is no effectively preventing medicine
Agent, can only rely on plantation disease-resistant variety based on comprehensive preventive health measures (simple osmanthus is good, Zou Yafei, Ma Cun. cotton verticillium wilt flows year after year
The reason for row and countermeasure. Cotton, 2003,30:13-14;Zhang Baolong, holds good, the Yang Yuwen of letter. verticillium wilt resistance of cotton by same study into
Exhibition .2012, Scientia Agricultura Sinica technology publishing house).But because verticillium wilt pathogenesis are complicated and cotton key resistance resource master
In there are the Wild ornamental resources such as Asiatic cotton and sea island cotton, to Disease Resistance Identification and breed breeding great obstacle is caused.Hold
It is easily caused progeny material and selects inaccurate, improved variety resistance is unstable, and the merit such as yield, fiber is difficult to be polymerized, seed selection
The problems such as inefficiency.Therefore, the molecular labeling with resistance to verticillium wilt close linkage is excavated, it is auxiliary using effectively reliability molecular labeling
Help breeding to accelerate breeding process, improve efficiency of selection.
With the development of cotton molecular marking technique, the structure of cotton high density genetic linkage mapses, obtain some with
The chain selected marker that can be used for molecular mark of resisting verticillium.Currently with the hybrid Population of upland cotton, and
Upland cotton and sea island cotton interspecific hybridization colony, have been obtained for the related QTL of more than 100 resistance to verticillium wilt, except the 6th, 10,12
It is external with 18 dyeing, these QTL be distributed on remaining all of chromosome (Bolek Y, El-Zik KM, Pepper AE,
Bell AA,Magill CW,et al.Mapping of verticillium wilt resistance genes in
cotton.Plant Sci,2005,168:1581–1590;Du WS,Du XM,Ma ZY(2004)Studies on SSR
markers of resistance gene of Verticillium wilt in cotton.J Northwest Sci
Tech UnivAgri and For,2004,3:20–24;Gao YQ,Nie YC,Zhang XL.QTL mapping of
genes resistant to Verticillium wilt in cotton.Cotton Sci,2003,2:73–78;Fang
H,Zhou HP,Sanogo S,Flynn R,Percy RG,et al.Quantitative trait locus mapping
for Verticillium wilt resistance in a backcross inbred line population of
cotton(Gossypiumhirsutum×Gossypiumbarbadense)based on RGA-AFLP
analysis.Euphytica,2013,194:79–91;Wang HM,Lin ZX,Zhang XL,Chen W,Guo XP,et
al.Mapping and quantitative trait loci analysis of Verticillium wilt
resistance genes in cotton.J Integr Plant Biol,2008,2:174–182;Yang C,Guo WZ,
Li GY,Gao F,Lin SS,et al.QTLs mapping for Verticillium wilt resistance at
seedling and maturity stages in Gossypiumbarbadense L.Plant Sci,2008,174:290–
298;Zhen R,Wang XF,Ma ZY,Zhang GY,Wang X.A SSR marker linked with the gene of
Verticillium wilt resistance in Gossypiumbarbadense.Cotton Sci,2006,5:269–
272;Jiang F,Zhao J,Zhuo L,Guo WZ,Zhang TZ.Molecular mapping of Verticillium
wilt resistance QTL clustered on chromosomes D7 and D9 in upland cotton.Sci
China Ser C(life Sci),2009,9:872–884;Ning ZY,Zhao R,Chen H,Ai NJ,Zhang X,et
al.Molecular tagging of a major quantitative trait locus for broad-spectrum
resistance to Verticillium wilt in upland cotton cultivar Prema.Crop Sci,
2013,53:2304–2312;Wang FR,Liu RZ,Wang LM,Zhang CY,Liu GD,et al.Molecular
marker of Verticillium resistance in upland cotton(Gossypiumhirsutum L.)
cultivar and their effects on assisted phenotypic selection.Cotton Sci,2007,
6:424–430;Yang C,Guo WZ,Zhang TZ.QTL mapping for resistance to Verticillium
wilt,fiber quality and yield traits in upland cotton(Gossypiumhirsutum L).Mol
Plant Breed,2007,5(6):797–805).These results of study show that cotton verticillium wilt resistance locus are positioned and verticillium wilt
Bacterium fungus strain it is related to cotton growth period (Wang et al., 2008;Yang et al.,2008;Yang et al.,2007).
Application of the molecular mark technology in terms of verticillium wilt resistance of cotton by same breeding is less, and main cause is verticillium wilt resistance of cotton by same machine
Reason is complicated, and it is stable that polyploid hybridization causes characters of progenies to be difficult to, and disease resistance trait identification stability and repeatability are general, cause seed selection
During easily occur that material is elected or superseded blindness is larger, and breeding parent and filial generation material qualification result it is inaccurate
Really.
Functional label is the one kind developed according to the polymorphism motif for causing phenotypic character to make a variation inside functional gene
New molecular marker.Because this kind of mark need not be verified further from intragenic functional motif, therefore this kind of mark
The presence or absence of target alleles just can be determined under different genetic background.Have in application compared with conventional genetic is marked
Bigger advantage.First, mark derives from target gene, isolates with it, it is possible to avoiding being produced because restructuring is exchanged
Select Error, colony target gene detect when it is more efficient;Further, since functional gene mark is directly from gene
The polymorphism motif of function is determined on site, once therefore functional label be developed, it is not necessary to by further checking just
Can apply under various different genetic backgrounds;It is effective to biological engineering colony and natural population;Secondly as being source
In gene internal, directly reflect objective trait performance, when carrying out the transfer of merit hence with backcross transformation, functional label
Application can preferably avoid Linkage drag, reduce importing (Andersen J R, L ü of the not beneficial gene of donor to acceptor
bberstedt T.Functional markers in plants.Trends Plant Sci,2003,8:554-560)。
Can be preferable according to the functional label of the invertase TaCwi-A1 of Grain Weight in Common Wheat key gene cell wall-bound exploitations
The size of thousand grain weight of wheat is distinguished on ground, can be used for molecular marker assisted selection (Xiang Jishan, Mu Peiyuan, Sang Wei, the Nie Ying of grain weight
It is refined, Xu Hongjun, Zhuan Li, Cui Fengjuan, Han Xinnian, Zou Bo. Grain Weight in Common Wheat gene TaCwi-A1 functional label CWI22, CWI21's tests
Card and application. Scientia Agricultura Sinica, 2014,47 (13):267-2679).According to paddy rice control mass of 1000 kernel gene TGW 6 with its etc.
The single base disappearance that position gene tgw 6 exists in functional area, the functional label for designing and filtering out the genes of TGW 6 can be accurate
Identify TGW 6 different genotype (Wang Jun, Yang Jie, Xu Xiang, Zhu Jinyan, Fan Fangjun, Li Wenqi, Wang Fangquan, Zhong Weigong. water
The development and utilization of the functional labels of rice mass of 1000 kernel gene TGW 6. rice in China science, 2014.28 (5):473-478).According to water
There is 8bp base deletions between rice recessiveness odor type kind and BAD2 genes (i.e. scent gene) sequence of non-odor type kind, design
Go out the InDel functional label GRFM04 of scent gene fgr, can exactly distinguish odor type homozygous genotype, non-odor type homozygosis
3 kinds of banding patterns of genotype and heterozygous genotypes, and the aroma characteristics performance corresponding with its plant or kind of 3 kinds of banding patterns is completely in one by one
Corresponding relation (Wang Feng, Li Jinhua, Liu Wuge, Liao Yilong, Zhu Manshan, Liu Zhenrong, Huang Huijun, Huang Dejuan. a kind of rice scent base
Because of the exploitation of functional label. rice in China science, 2008,22 (4):347-352).
The Ve genes of tomato (Lycopersiconesculentum) be first clone resisting verticillium gene, Ve genes
Site includes two genes of Ve1 and Ve2, is a kind of receptor protein (receptor-like protein, RLP) gene, by its turn
To enter be improved after susceptible Potato Cultivars the resistance to verticilliumalbo-atrum (Kawchuk LM, Hachey J, Lynch DR, et
al.Tomato Ve disease resistance genes encode cell surface-like
receptors.Proceedings of the national academy of sciences of the united
states of America,2001,98:6511-6515.).Cotton GbVe gene families are equally extremely closed to the resistance of verticillium wilt
It is important.Zhang etc. (2011) has cloned GbVe genes from sea island cotton Pima90-53, and the gene falls within RLP GFPs,
55.9% and 57.4% is respectively with tomato Ve1 and Ve2 gene similitude, the resistance to verticillium wilt of the gene transformation of Arabidopsis thaliana strain shows
Write and improve (Zhang, Y., Wang, X., Yang, S., Chi, J., Zhang, G.and Ma, Z.Cloning and
characterization of a Verticillium wilt resistance gene from
Gossypiumbarbadense and functional analysis in Arabidopsis thaliana.Plant
Cell Rep 30(2011),pp.2085-2096.);Zhang etc. (2012) is cloned from the cotton variety H7124 of resisting verticillium island
With tomato Ve1 genes (Fradin, E.F., Zhang, Z., Juarez Ayala, J.C., Castroverde, C.D.,
Nazar,R.N.,Robb,J.,Liu,C.M.and Thomma,B.P.Genetic dissection of Verticillium
Wilt resistance mediated by tomato Ve1.Plant Physiol 150 (2009), pp.320-332.) and
The Gbve1 genes of cotton GbVe genes (Zhang et al.2011) homeologous, the gene also has typical plant disease-resistant
Protein structure domain, the homology with tomato Ve1 genes (Fradin et al.2009) is 51%, with cotton GbVe genes
The homology of (Zhang et al.2011) is 69%.Using VIGS silences Gbve1 gene sea island cotton H7124 can be made to verticillium wilt
Resistance lose;Also, the result of Gbve1 genetic transformation arabidopsis and upland cotton all shows Gbve1 gene pairs High pathogenicities
Defoliation and non-defoliation verticillium wilt pathogen all have resistance (Zhang, B., Yang, Y., Chen, T., Yu, W., Liu, T., Li,
H.,Fan,X.,Ren,Y.,Shen,D.,Liu,L.,Dou,D.and Chang,Y.Island cotton Gbve1 gene
encoding a receptor-like protein confers resistance to both defoliating and
non-defoliating isolates of Verticilliumdahliae.PLoS One 7(2012a),
pp.e51091.).Yang etc. (2014) has cloned same with tomato Ve1 Gene Partials from the cotton variety H7124 of resisting verticillium island
The Gbvdr5 genes in source, the arabidopsis of the gene and Cotton Transformation strain equally have defoliation and non-defoliation resistance to verticillium wilt
(Yang YW,Ling XT,Chen TZ,CaiLiW,Liu TL,Wang JY,Fan XH,Ren YZ,Hongbo Yuan,Zhu
W,Zhang BL,Ma Din-Pow.A Cotton Gbvdr5 Gene Encoding a Leucine-Rich-Repeat
Receptor-Like Protein Confers Resistance to Verticilliumdahliae in Transgenic
Arabidopsis and Upland Cotton.Plant Molecular Biology Reporter,2014,DOI
10.1007/s11105-014-0810-5)。
This research develops a kind of cotton verticillium wilt functional gene mark according to cotton GbVe gene families, and proves the mark
Note can accurately detect the anti-sense material of verticillium wilt, can be applicable to screen cotton hybrid introgressive line plant, improve Breeding Efficiency, show
Write and save breeding screening cost.
3rd, the content of the invention
Technical problem
The purpose of the present invention is:One cotton verticillium wilt functional gene mark is provided and its is applied, this labeled fragment is present
In the susceptible material of cotton verticillium wilt, and lack in disease-resistant material or be mutated.The fragment is dominant molecular labeling.Can utilize
The mark accurately detects the resistance to verticillium wilt of different cotton varieties or material, can be applicable to screen the plant of cotton hybrid introgressive line
Strain, improves Breeding Efficiency, significantly saves breeding screening cost, obtains the preferable material of resistance to verticillium wilt.
Technical scheme
One cotton verticillium wilt functional gene mark and its application, it is characterised in that:
Use labeled primer Gbvds1549:
Left end primer sequence is:TTGAGCAATTGACAAGAATAGAGCT
Right-hand member primer sequence is:TAGTAATATTGGTCATTATTTGC.
With the disease-resistant material of labeled primer Gbvds1549 amplification cotton verticillium wilts or kind DNA, without amplified production.And expand
Sensitizing disease material or kind DNA, the amplified fragments of acquisition are 1549bp, as cotton verticillium wilt functional gene mark
Gbvds1549.This labeled fragment is lacked in disease-resistant material or is mutated in the susceptible material of cotton verticillium wilt.The fragment is
Dominant molecular labeling.
The cotton verticillium wilt functional gene flag sequence, is one of following nucleotide sequence:
1) DNA sequence dna in sequence table shown in SEQ ID NO.1;
2) nucleotide sequence of the DNA sequence dna hybridization that can be limited with SEQ ID NO.1 in sequence table.
Described flag sequence, it is characterised in that the bar of the DNA sequence dna hybridization limited with SEQ ID NO.1 in sequence table
Part is in 0.1 × SSPE or 0.1 × SSC, the solution of 0.1%SDS, under the conditions of 65 DEG C film to be washed.
Described flag sequence, it is characterised in that:Nonreactive of the molecule labelled series from one excalation of cotton
Property effect surface receptor protein gene ghvdr6, the gene nucleotide length is 2121bp, and the amino acid length of coding is
706。
Beneficial effect
1st, present invention obtains a cotton verticillium wilt functional gene marks Gbvds1549.The present invention is sequenced by BAC,
Obtain a surface receptor protein gene ghvdr6 without entire reading frame.Resisted using there is verticillium wilt known to the gene and other
Property effect gene order differential position design gene specific primer, the primer expands susceptible material or kind DNA, the expansion of acquisition
Increasing fragment is 1549bp, nucleotide sequence as shown in SEQIDNO.1, but with the primer expand the disease-resistant material of cotton verticillium wilt or
Kind DNA, without amplified production.And the method for developing Functional marker on this basis.By detecting the functional molecular
Mark, can accurately detect the resistance to verticillium wilt of different cotton varieties or material, can be applicable to screen cotton hybrid introgressive line
Plant, improves Breeding Efficiency, significantly saves breeding screening cost, obtains the preferable material of resistance to verticillium wilt.
2nd, Gbvds1549 is the functional gene mark of the cotton verticillium wilt of first report.The present invention obtain mark be
Functional gene is marked, with obvious advantage compared with the genetic marker reported before.Functional gene mark is according to function base
Because inside causes a kind of New molecular marker for developing of polymorphism motif that phenotypic character makes a variation.Due to it is this kind of mark from
Intragenic functional motif, therefore this kind of mark need not further verify and just can determine mesh under different genetic background
The presence or absence of mark allele.Gbvds1549 is according to from the surface receptor protein closely related with cotton verticillium wilt resistance
Gene, but the gene is one to be lacked, nonfunctional gene.So the mark is existed only in susceptible material.
3rd, the Breeding Efficiency of resistance to verticillium wilt material is improved, it is cost-effective.Traditional genetic marker receives genetic background, plant
Growth period and environmental condition etc. affect.Such as cotton verticillium wilt mark is different because of verticillium wilt pathogen fungus strain and cotton growth period,
The related QTL of more than 100 resistance to verticillium wilt is had been obtained at present, except the 6th, 10,12 and 18 articles of dyeing are external, is distributed in it
On remaining all of chromosome.These marks are difficult to be effectively applied to molecular mark.And functional label is from control
The gene of proterties processed itself, can use in different genetic background and breeding time.
4th, the present invention contributes to cloning verticillium wilt disease-resistant gene and understanding its resistance mechanism.Gbvds1549 is according to source
In the surface receptor protein gene closely related with cotton verticillium wilt resistance, but the gene is one to be lacked, nonfunctional base
Cause.The homologous gene in the site in by cloning disease-resistant material, it is possible to be separated to one and resistance to verticillium wilt is played a crucial role
Disease-resistant gene.
4th, illustrate
The nucleotides and amino acid sequence of Fig. 1 ghvdr6.
The nucleotides of Fig. 2 ghvdr6 genes similar with other known cotton acceptors compares.Black surround part be ghvdr6 and its
The larger position of his gene difference, i.e. Gbvds1549 labeled primers position.
Amplification of Fig. 3 Gbvds1549 labeled primers in the anti-sense material of difference.
5th, specific embodiment
Method therefor is if no special instructions conventional method in following embodiments, and the primer sequence is by Shanghai English
Pretty Bioisystech Co., Ltd's synthesis, the percentage composition is weight/mass percentage composition.Resistance Strain of Cotton sense material used is public affairs
Know public, pertinent literature and information are shown in Table 1.
(1) exploitation of cotton Gbvds1549 marks
According to one section of cotton est sequence (accession number in Gene Index:TC121084) design primer 5 '-
TTCTGGTCCAATACCATCATTCT-3 ', 5 '-CTTAGATTCAGTACTCCAAGAGA-3 ' expand upland cotton DNA profiling, obtain
To about 1Kb or so bands, by the sequencing fragment, its similarity for there was only 76% with original EST fragments, the fragment coding are found
1 brand-new cotton surfaces receptor protein gene, is further somebody's turn to do according to the fragment design primer by chromosome walking
The full length sequence of gene, is GhVdr2 (patents by the unnamed gene:Cotton verticillium wilt disease-resistant related gene GhVdr2 and its should
With grant number:ZL 201110066390.9).
Because this kind of surface receptor protein gene has many similar sequences in genome, in order to obtain more this kind of bases
Cause, according to GhVdr2 primer is designed
GhVdr2-F595:5 '-CTTGATGGGGTGAATATTAGAGCA-3 ',
GhVdr2-R1021:5’-ATTGCCCAAGGTTACCGATAGAAT-3’
Amplification upland cotton DNA profiling.Cotton BAC (bacterial artificial chromosomes are screened with the about 400bp fragments for obtaining as probe
Body group) library (it is maxxa to build cotton variety used by library, and library derives from Clemson University), it is obtained 40
Individual BAC positive colonies.
BAC clone D1 are sequenced, commission Shanghai Han Yu biotech companies are carried out.Sequencing result shows, the clone
In addition to containing the surface receptor protein gene with entire reading frame, such protein gene sequence of also one disappearance should
Gene nucleotide is 2121bp, and the amino acid length of coding is only 706, is ghvdr6 (Fig. 1) by the unnamed gene.And tomato
Resistance to verticillium wilt gene Ve1 length is that (Fradin et al., 2009), cotton Gbve is 3819bp (Zhang et to 3162bp
Al., 2011), Gbve1 is that (Zhang et al., 2012), Gbvdr5 is 3454bp (Yang et al.2014) to 3417bp.Profit
Ghvdr6 and the resistance to verticillium wilt gene order (Fig. 2) reported are analyzed with DNAMAN.9 genes are devised for diversity sequence
Special primer, for expanding the DNA of the anti-sense material of verticillium wilt.
(2) molecular markers for identification of the anti-sense material of verticillium wilt
Identified using the anti-sense material of 30 verticillium wilt (be public, be shown in Table 1 bibliography), wherein disease-resistant material
21, material, susceptible material 9 (table 1).These are all to be presently considered to the preferably or extremely susceptible cotton material of resistance, and often
It is used as parent and builds segregating population.Using CTAB methods extract cotton genomic dna (PATERSON A H, Brubaker C L,
Wendel J F.A rapid methodfor extraction cotton (spp.)genomic DNA suitablefor
RFLP or PCR analysis.Plant MolBiol Rep,1993,11:122-127).Using 9 gene specifics of design
Primer expands the DNA of the anti-sense material of verticillium wilt, and PCR reaction systems are:The μ l of DNA profiling 1, primer are added in 25 μ l reaction systems
Each 5nmol, 5 μ l5 × primeSTAR buffer (Mg2+Plus) PCR buffer solutions, 0.2mMdNTP, 1U primeSTARHS DNA
Polymerase (TaKaRa companies) enters performing PCR amplification.PCR amplification conditions are:94 DEG C 45 after 94 DEG C of 3' ", 56 DEG C 45 ", 72 DEG C
1'30 ", circulates 36 times, then 72 DEG C of extension 10'.PCR primer is detected on 1% Ago-Gel.As a result only a pair are shown
Primer amplify in all of susceptible material length be 1549bp fragment, nucleotide sequence as shown in SEQIDNO.1, but
Without amplified production (Fig. 3) in disease-resistant material.The mark is named as into Gbvds1549, its labeled primer is:
Left end primer sequence is:TTGAGCAATTGACAAGAATAGAGCT
Right-hand member primer sequence is:TAGTAATATTGGTCATTATTTGC.
Table 1. is used for the anti-sense material of 30 verticillium wilt of molecular markers for identification
SEQ ID NO.1:
TTGAGCAATTGACAAGAATAGAGCTTGCGACTTGCAATTTCAGTGGAGCCATACCCAAAACAATGAAGA
AACTTACCCAACTTGTGTATCTGGATTTTTCCTTTAACCGTTTTTCTGGTCCAATACCATCATTCTCATCAGCCAGA
AATCTTATATACCTAAGCCTTAGTTATAATCAGTTAAATGGTGGAATTCATTCCACTGATTGGTCAAGTCTTTCTAA
GCTAGAAATTGTTTACTTAGGAAACAACAAGTTAAGTGGAACCATTCCACCGGCTTTGTTTTGCATTCCATCACTGC
GTGGACTTCACCCTTATCAAAACCAATTCAAGGGTAACCTTAATGACCTTCATGGTAAGGCCTCTTTATTGCTTGAG
GACCTTGATCTTAGTAGCAACAAGTTACAAGGGCAATTCCCAATGTCTATGTTTGAACTCCATGGTCTGAAGTTGCT
ATCCCTTTCTTCAAACAACTACAGTGGCTCGATACCAATGAGTGCCTTTCAGAACTTGAGGAATCTTTCTTACCTTG
ATCTCTCATATAACAGGTTGTCTATTGATGCCACTGATACTAATATTTCCTCACTTTCTTTCCCTACCATCGTCACA
TTGAAGTTGACATCTTGCAACTTAACGGAGTTCCCTGATTTCTTGAAATATCAGTCTAGATTATCATATCTAGACCT
TTCAAACAACCAGATTCAAGGGAAAATACCGAATTGGATTTGGAAAGTGAGAAGCCTTGGACACCTAAATCTTTCTC
AAAACTTCATTGTAGAATTTGATAAATCTTTGAAGAATATAAATTCTACTCTCAATGTTTTGGACCTGCATGGCAAT
CAATTGCAAGGGCAAATCCAAATTCTTCCACCATATGTCACTTATTTGGATTACTCAAACAACAATTTCAGCTCTGT
TTTACCAGCTCAGCTCGGTGACTTCCTCCAGTTTGCTTATTTCTTCTCTGTCTCAGGCAATAACTTCAATGGGAGTA
TTCCCAAGTCGATATGCAGTAGCTTATATCTCAAAGTACTTGATATGTCTTATAATTACTTGAGTGGGTCAATTCCT
AAATGCCTGACTCAAATGAGTGCATCTCTTGGAGTACTGAATCTAAAGGGAAACAACCTCAGTGGCATCATTTCCGA
CGCTTTTCCAGAAAGTTGTAAGTTACAAACTCTAGATCTCAATCAGAACCGATTGGAAGGAAAGGTTCCAGAATCAT
TGGGGAATTGCAAAGAGCTGGAGGTTGTAGACATTGGCAACAATCAGATCAGTGGCAGCTTCCCATGCCATTTGGAG
AATATATCCAAGTTGCGTGTCCTTGTTTTACGATCTAACAAATTCAACGGCAGTATTCATTGTCACAAGAACAATAC
CAGCTGGCCAATGCTTCAGATTGTTGACTTAGCATCCAACAATTTTAGTGGTAAACTGCATCTAACTGGTTTGGGGA
CCTGGGAGGCTATGCGGCCTAATCATGATAAAAACCAATCAGAGCTCCAACATCTCATGTTTGAGGTCTTAGCAAAT
AATGACCAATATTACTA
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>A kind of molecular labeling of cotton verticillium wilt functional gene and its application
<130> 0
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 1549
<212> DNA
<213>Manually
<220>
<221> Gbvds1549
<222> (1)..(1549)
<223>
<400> 1
ttgagcaatt gacaagaata gagcttgcga cttgcaattt cagtggagcc atacccaaaa 60
caatgaagaa acttacccaa cttgtgtatc tggatttttc ctttaaccgt ttttctggtc 120
caataccatc attctcatca gccagaaatc ttatatacct aagccttagt tataatcagt 180
taaatggtgg aattcattcc actgattggt caagtctttc taagctagaa attgtttact 240
taggaaacaa caagttaagt ggaaccattc caccggcttt gttttgcatt ccatcactgc 300
gtggacttca cccttatcaa aaccaattca agggtaacct taatgacctt catggtaagg 360
cctctttatt gcttgaggac cttgatctta gtagcaacaa gttacaaggg caattcccaa 420
tgtctatgtt tgaactccat ggtctgaagt tgctatccct ttcttcaaac aactacagtg 480
gctcgatacc aatgagtgcc tttcagaact tgaggaatct ttcttacctt gatctctcat 540
ataacaggtt gtctattgat gccactgata ctaatatttc ctcactttct ttccctacca 600
tcgtcacatt gaagttgaca tcttgcaact taacggagtt ccctgatttc ttgaaatatc 660
agtctagatt atcatatcta gacctttcaa acaaccagat tcaagggaaa ataccgaatt 720
ggatttggaa agtgagaagc cttggacacc taaatctttc tcaaaacttc attgtagaat 780
ttgataaatc tttgaagaat ataaattcta ctctcaatgt tttggacctg catggcaatc 840
aattgcaagg gcaaatccaa attcttccac catatgtcac ttatttggat tactcaaaca 900
acaatttcag ctctgtttta ccagctcagc tcggtgactt cctccagttt gcttatttct 960
tctctgtctc aggcaataac ttcaatggga gtattcccaa gtcgatatgc agtagcttat 1020
atctcaaagt acttgatatg tcttataatt acttgagtgg gtcaattcct aaatgcctga 1080
ctcaaatgag tgcatctctt ggagtactga atctaaaggg aaacaacctc agtggcatca 1140
tttccgacgc ttttccagaa agttgtaagt tacaaactct agatctcaat cagaaccgat 1200
tggaaggaaa ggttccagaa tcattgggga attgcaaaga gctggaggtt gtagacattg 1260
gcaacaatca gatcagtggc agcttcccat gccatttgga gaatatatcc aagttgcgtg 1320
tccttgtttt acgatctaac aaattcaacg gcagtattca ttgtcacaag aacaatacca 1380
gctggccaat gcttcagatt gttgacttag catccaacaa ttttagtggt aaactgcatc 1440
taactggttt ggggacctgg gaggctatgc ggcctaatca tgataaaaac caatcagagc 1500
tccaacatct catgtttgag gtcttagcaa ataatgacca atattacta 1549
<210> 2
<211> 25
<212> DNA
<213>Manually
<220>
<221>Primer Gbvds1549 is left
<222> (1)..(25)
<223>
<400> 2
ttgagcaatt gacaagaata gagct 25
<210> 3
<211> 23
<212> DNA
<213>People
<220>
<221>Primer Gbvds1549 is right
<222> (1)..(23)
<223>
<400> 3
tagtaatatt ggtcattatt tgc 23
Claims (1)
1. a kind of application of the molecular labeling of cotton verticillium wilt functional gene, it is characterised in that:
Use molecular labeling primer Gbvds1549:
Left end primer sequence is:TTGAGCAATTGACAAGAATAGAGCT
Right-hand member primer sequence is:TAGTAATATTGGTCATTATTTGC
The disease-resistant material of amplification cotton verticillium wilt or kind DNA, without amplified production;And susceptible material or kind DNA are expanded, obtain
Amplified fragments be 1549bp, this labeled fragment is present in the susceptible material of cotton verticillium wilt, and in disease-resistant material disappearance or
Mutation, the fragment is dominant molecular labeling.
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CN101235417B (en) * | 2007-01-30 | 2011-01-05 | 广东海洋大学 | Molecule marking method based on functional gene polymorphism |
WO2012083547A1 (en) * | 2010-12-23 | 2012-06-28 | Zuo Kaijing | Anti-verticillium wilt gene of gossypium barbadense and its use |
CN102229938B (en) * | 2011-06-17 | 2012-09-26 | 江苏省农业科学院 | Gene Gbvdr5 giving verticillium resistance in plants and use thereof |
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