CN101899437A - Functional molecular marker for resistance identification of blight in Cucumis melo L. and application thereof - Google Patents
Functional molecular marker for resistance identification of blight in Cucumis melo L. and application thereof Download PDFInfo
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Abstract
The invention discloses a functional molecular marker for the resistance identification of blight in Cucumis melo L. The CAPS (cleaved amplified polymorphic sequences) primer sequence in the invention is any one of the followings: forward primer 5'-TGCTCCTTTGGCTTCCTGT-3', reverse primer 5'-GAGTCTATTGTTTCGCTTCA-3'; forward primer 5'-GGAAGTGAGGTGTTGAATT-3', reverse primer 5'-TACACATTGGTCCGTTAGAC-3'; or forward primer 5'-AGACGTAGCATTGCTTCTCTAG-3', reverse primer 5'-TGGCATCCTTCAGCACCTTC-3'; or, the AS-PCR (Allele specific polymerase chain reaction) primer sequence in the invention is as follows: forward primer 5'-GTGTAACACAAATTCCTCAACA-3', reverse primer 5'-GACGTAGCATTGCTTCTGTAGA-3'. The functional molecular marker is applicable to molecular marker-assisted breeding of the blight in Cucumis melo L. or identification of the resistance genes of the blight in Cucumis melo L.
Description
Technical field
The invention belongs to vegetable disease-resistant molecular marker breeding technical field, be specifically related to a kind of muskmelon fusarium wilt disease resistance gene Fom-2 functional molecular marker and application thereof, thereby provide a kind of novel molecular mark for the marker assisted selection of muskmelon fusarium wilt disease resistance.
Background technology
Muskmelon (Cocumis melo L.) has higher commerce product and is worth and economic worth as a kind of important garden crop in Curcurbitaceae (Cucurbitaceae) plant, is one of important cash crop of China.Along with the continuous expansion of commercialization of muskmelon idioplasm resource and cultivated area, disease and pest takes place serious day by day in recent years, forces to produce to go up a large amount of chemical reagent that use, and has had a strong impact on the yield and quality (Sherf and MacNab 1986) of muskmelon.Wherein the muskmelon blight is a destructive soil-borne disease in a kind of world wide that is caused by Fusarium oxysporum, causes financial loss (the Leach JG1938 of muskmelon more than 90%; Martyn RD 1987).Identified at present to separate to obtain 4 kinds of wilt physiological strains, be respectively physiological strain 0,1,2 and 1-2.Genetic analysis shows, 2 independently gene Fom-1 and Fom-2 control resistance (Risser et al.1976 to physiological strain 0,2 and 0,1 respectively; Risser and Mas 1965; Robinson et al.1976).Wherein the Fom-2 gene obtains separating (Joobeur et al.2004) by the method for map based cloning.The Fom-2 gene belongs to the R gene of NBS-LRR class, and this genoid coding NBS (nucleotide binding site, NBS) and LRR (leucine-richrepeat, LRR) two structural domains of relatively guarding.Usually plant specially to change resistance be because the identification of the nontoxic gene that host's antagonism albumen produces, and then cause a signal cascade reaction and defensive raction (Hammond Kosack and Jones 1997; Martin et al.2003).The R gene of plant constantly changes discerns the nontoxic gene that produces variation, and this helps, and people find on molecular level and the knowledge of natural environment is selected.Now people from many species the clone obtain the R gene, and verified that they are at the resistance and the functional diversity of anti-storeroom not.There is the polymorphism of sequence in many Arabidopis thaliana and other several species R genes of studies show that, this all or plant in influence the nucleotide sequence variation of phenotype identification help the development (Andersen and Lubberstedt, 2003) of functional molecular marker.
Cultivating good anti-blight melon variety at present becomes one of effective way that solves the withered disease problem of muskmelon, and the development of molecule assisted Selection (MAS) means can be shortened muskmelon fusarium wilt disease resistance breeding process greatly.But there are certain genetic distance in employed molecule marker and target gene in the breeding for disease resistance process at present, and easily the generation reorganization exchanges and separates, thus the accuracy that influence is selected.
Functional molecular marker is the novel molecular mark that functional mononucleotide polymorphism site exploitation forms in the functional gene motif with phenotypic correlation, and the exploitation of functional molecular marker at first needs to identify in the colony sequence with the functional motif of the functional gene of phenotypic correlation.AS-PCR and CAPS method have been successfully applied to exploitation (the Chiapparino et al.2004 based on a series of functional molecular markers of SNPs; Kim et al.2006; Yeam et al.2005).Functional molecular marker does not need the plant and instrument or the complex operating steps of complex and expensive, therefore is fit to very much be applied to the molecular breeding process.Along with increasing functional gene and allelic separation and note, functional molecular marker has become a class novel molecular mark, can improve the efficient of mark greatly.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of and the relevant functional molecular marker of muskmelon fusarium wilt disease resistance gene Fom-2, and it is applicable to the assisted Selection of the anti-blight of muskmelon.
In order to solve the problems of the technologies described above, the invention provides a kind of functional molecular marker that the muskmelon fusarium wilt disease resistance is identified that is used for,
The CAPS primer sequence that this molecule marker adopted be following any one:
CAPS1: forward primer is 5 '-TGCTCCTTTGGCTTCCTGT-3 ' (SEQ ID NO:3)
Reverse primer is 5 '-GAGTCTATTGTTTCGCTTCA-3 ' (SEQ ID NO:4),
CAPS2: forward primer is 5 '-GGAAGTGAGGTGTTGAATT-3 ' (SEQ ID NO:5)
Reverse primer is 5 '-TACACATTGGTCCGTTAGAC-3 ' (SEQ ID NO:6),
CAPS3: forward primer is 5 '-AGACGTAGCATTGCTTCTCTAG-3 ' (SEQ ID NO:7)
Reverse primer is 5 '-TGGCATCCTTCAGCACCTTC-3 ' (SEQ ID NO:8);
Perhaps, the AS-PCR primer sequence that this molecule marker adopted is:
AS-PCR: forward primer is 5 '-GTGTAACACAAATTCCTCAACA-3 ' (SEQ ID NO:9)
Reverse primer is 5 '-GACGTAGCATTGCTTCTGTAGA-3 ' (SEQ ID NO:10).
The present invention also provides the purposes of above-mentioned functions molecule marker simultaneously: be used for the marker assisted selection of muskmelon blight, perhaps be used to identify muskmelon fusarium wilt disease resistance gene.
The contriver has found that muskmelon fusarium wilt disease resistance gene Fom-2 has 3 SNP sites in the LRR zone of disease-resistant and susceptible germplasm, and the nucleotide sequence of muskmelon fusarium wilt disease resistance gene Fom-2 is as described in the sequence table SEQ ID NO:1.Fusarium wilt disease resistance and susceptible material have the base mutation of an A-G at the 281bp place of sequence table SEQ ID NO:1, the base mutation of an A-G is arranged at the 1075bp place, and the base mutation (Fig. 1) of a T-C is arranged at the 1216bp place.Nucleotide sequence after the sudden change is as described in the sequence table SEQ ID NO:2.
SNP feature according to the sequence of above-mentioned SEQ ID NO:1 designs primer respectively, exploitation FMs, and the primer of described CAPS and AS-PCR method is to being respectively:
CAPS 1: forward primer is 5 '-TGTTTGGAGAAAGTTGAAAGTC-3 '
Reverse primer is 5 '-ACCATACAAACCTATCTCTATT-3 ';
CAPS2: forward primer is 5 '-GGAAGTGAGGTGTTGAATT-3 '
Reverse primer is 5 '-TACACATTGGTCCGTTAGAC-3 ';
CAPS3: forward primer is 5 '-AGACGTAGCATTGCTTCTCTAG-3 '
Reverse primer is 5 '-TGGCATCCTTCAGCACCTTC-3 '.
AS-PCR: forward primer is 5 '-GTGTAACACAAATTCCTCAACA-3 '
Reverse primer is 5 '-GACGTAGCATTGCTTCTGTAGA-3 '.
This CAPS and AS-PCR primer are to successfully identifying the disease-resistant and susceptible germplasm of muskmelon (Fig. 2,3), and be monogenic inheritance law of segregation (table 1,2) at F2 from generation to generation, use above-mentioned two kinds of methods and can successfully identify the genotype (table 3) of muskmelon idioplasm fusarium wilt disease resistance.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is the disease-resistant and susceptible germplasm Fom-2 of muskmelon of the present invention gene LRR region S NP site figure;
Among Fig. 1: Fom-2-R represents blight disease-resistant gene type, and Fom-2-S represents the susceptible genotype of blight;
Fig. 2 is that the CAPS method is identified the disease-resistant and susceptible germplasm comparison diagram of muskmelon;
Among Fig. 2: FF is disease-resistant homozygous genotype, and Ff is disease-resistant heterozygous genes type, and ff is susceptible homozygous genotype, and M is a dna molecular amount standard;
Fig. 3 is that the AS-PCR method is identified the disease-resistant and susceptible germplasm comparison diagram of muskmelon;
Among Fig. 3: FF is disease-resistant homozygous genotype, and Ff is disease-resistant heterozygous genes type, and ff is susceptible homozygous genotype, and M is a dna molecular amount standard.
Embodiment
Concrete steps are as follows:
(1) find the sequence information of muskmelon fusarium wilt disease resistance gene Fom-2 in ncbi database (http://www.ncbi.nlm.nih.gov/), Fom-2 gene accession number is AY583855.The Fom-2 gene belongs to the R gene of NBS-LRR class, two relatively more conservative structural domains of coding NBS and LRR.
(2) design primer and synthetic, at two ends, Fom-2 gene LRR district design primer, primer sequence is as follows:
Fom-2: forward primer: GAGTCTATTGTTTCGCTTC
Reverse primer: TGCTCGTCTCTGGGTCACCTTC.
(3) extraction of genomic dna
(a) get disease-resistant (Cantaloupe, high resistance to wilt good material) of the above-mentioned muskmelon of 0.5g and susceptible germplasm (celestial fruit 027-5, high sense blight material), F respectively
1, F
2Different muskmelon idioplasm tender leafs in mortar, the liquid nitrogen state grinds down, adds the 2 * CTAB (30 μ l mercaptoethanol mixing) of 65 ℃ of preheatings of 600 μ l, transfers in the centrifuge tube 65 ℃ of insulations 1 hour.
(b) chloroform of adding 600 μ l: primary isoamyl alcohol (24/1 volume ratio) shakes up gently, and 4 ℃, 13000rpm is centrifugal, 10 minutes.
(c) get supernatant, repeat (b) step once.
(d) supernatant of getting step (c) gained adds and the isopyknic Virahol of supernatant in another centrifuge tube, puts upside down mixing, leaves standstill 13000rpm, centrifugal 10 minutes 5 minutes.
(e) remove supernatant, add 700 μ l Wash Buffer rinsing DNA precipitation, centrifugal, repeat once.
(f) add 15 μ lTE-Rnase, 37 ℃ are incubated 1 hour.
(g) add 2mol/L NaCl 100 μ l+100% ethanol 250 μ l mixings ,-20 ℃ leave standstill 30min, centrifugal 10 minutes of 13000rpm.
(h) remove supernatant, add 500 μ l 70% (volume ratio) ethanol, ethanol is removed in rinsing, repeats once, and normal temperature dries.
(i) add 30-50 μ l ddH
2O or TE, Hui Rong ,-20 ℃ of preservations are standby.
Annotate: 2 * CTAB prescription:
CTAB： 4g
1M?Tris-HCl(pH8.0)： 20ml
EDTA： 1.488g
NaCl： 16.352g
Use earlier 70ml ddH
2O dissolving is settled to the 200ml sterilization again, cooling back 0.2-1%2-mercaptoethanol (400 μ l) chloroform-primary isoamyl alcohol (24: 1): add the 96ml chloroform earlier, add the 4ml primary isoamyl alcohol again, shake up and get final product.
TE-Rnase:15 μ l TE adds the Rnase of 1/100 volume.
Wash Buffer:10mmol/L acetate ammonia
75% (volume ratio) ethanol
The reagent and the condition of (4) chain polymerization enzyme reaction (PCR):
At first following reagent is mixed in together
10 * Taq dna polymerase buffer liquid, 5 μ l
Forward primer (1.25 μ g/l) 0.4 μ l
Reverse primer (1.25 μ g/l) 0.4 μ l
Deoxynucleoside acid mixture (dNTP) 1 μ l
Taq archaeal dna polymerase 0.4 μ l
Aqua sterilisa 41.8 μ l
Cumulative volume 50 μ l
The PCR reaction conditions is: behind 94 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 50 ℃ of annealing 45s, 72 ℃ are extended 1.5min, and 35 circulations are extended 10min for back 72 ℃.The PCR product carries out 1% agarose gel electrophoresis and detects.The purpose band is carried out cloning and sequencing, and concrete grammar is as follows:
(a) ligation, centrifuge tube add following each component in proper order:
H
2O： 2μl
T
4DNA?Ligase?10×Buffer： 1μl
PUCM-T carrier: 1 μ l
PCR product: 4 μ l
PEG4000： 1μl
T
4DNA?Ligase： 1μl；
(b) preparation (CaCl of competent cell
2Legal system is equipped with competent cell):
1. the single bacterium colony of the new activatory E.coli of picking DH5 α is inoculated in the 5ml LB liquid nutrient medium, about 37 ℃ of following shaking culture 12h, until the logarithmic growth later stage.
2. with bacterium liquid with 1: 100-1: 150 ratio (weight ratio) is inoculated in the 100ml LB liquid nutrient medium, about 37 ℃ of following shaking culture 2h, to OD600=0.5.
3. nutrient solution is changed in the centrifuge tube, place 10min on ice, then in 4 ℃ of centrifugal 10min of following 10000rpm.
4. abandon supernatant, with the CaCl2 solution 30ml of the 0.1mol/L of precooling suspension cell gently, place 10min on ice after, 4 ℃ of centrifugal 10min of following 10000rpm.Repetitive operation once.
5. abandon supernatant, add the CaCl of the 0.1mol/L that contains 15% (volume ratio) glycerine of 3ml precooling
2Solution is suspension cell gently, place 5min on ice after, competent cell, be distributed into the aliquot of 200 μ l, place-70 ℃ of preservations standby.
(c) heat shock method transformed into escherichia coli DH5 α:
1. get the competent cell suspension 200 μ l of above-mentioned (b) step preparation, shake up the connection product that the back adds 4 μ l above-mentioned (a) step, blow and beat evenly ice bath 30 minutes with the rifle head gently;
2. 42 ℃ are incubated 90 seconds, put into ice rapidly, ice bath 5 minutes;
3. the 1ml LB liquid nutrient medium (Amp that adds 37 ℃ of preheatings
-), mixing, 37 ℃ are incubated 1 hour, make cellular-restoring normal growth state;
4. 3000rpm is centrifugal 10 minutes;
5. supernatant discarded, 200 remaining μ l blow and beat evenly gently with the rifle head, evenly coat the LB that contains 15 μ l X-Gal (20mg/ml) and 100 μ l IPTG (24mg/ml) after bacterium is fully suspended to screen (Amp on the plate culture medium
+);
6. 37 ℃ face up place half hour after, be inverted and cultivated 8-12 hour, observe.
The LB culture medium prescription:
Add following each component in the 100ml system:
Peptone (Typtone): 1g
Yeast extract (Yeast Extract): 0.5g
NaCl： 1g
NaOH(1mol/L)： 100μl
Use earlier 70ml ddH
2O dissolving is settled to 100ml again, the high pressure-temperature back 4 ℃ of preservations of sterilizing.
(5) plasmid purification:
(a) in Bechtop, get single white colony, be inoculated in the 5mlLB nutrient solution that contains 50 μ g/ml Amp 37 ℃ of overnight incubation with aseptic rifle choicest.
(b) get 1-2ml bacterium liquid in the Epeendorf pipe, centrifugal 1 minute of 12000rpm;
(c) add 100 μ l Solution I, with the abundant suspension bacteria liquid of rifle head;
(d) add 200 μ l SolutionII, put upside down the Epeendorf pipe, ice bath 3 minutes;
(e) the Solution III of adding 150 μ l precoolings puts upside down the Epeendorf pipe, ice bath 5 minutes;
(f) 4 ℃, centrifugal 5 minutes of 12000rpm, supernatant liquor adds isopyknic phenol/chloroform mixing;
(g) 4 ℃, centrifugal 5 minutes of 12000rpm, supernatant liquor adds 2 times of volume of ethanol, and room temperature was placed 5 minutes;
(h) 4 ℃, centrifugal 5 minutes of 12000rpm, with 70% ethanol rinsing once, and the airing precipitation, 50 μ l TE melt precipitation, and-20 ℃ of preservations are standby.
Wherein:
Solution I:100ml system:
1mol/L?Tris-HCl(pH8.0)： 2.5ml
0.5mol/L?EDTA(pH8.0)： 2.0ml
20％Glucose(1.11mol/L)： 4.5ml
dH
2O： 91ml
4 ℃ of preservations behind the autoclave sterilization.
Solution II:100ml system:
10％SDS： 10ml
2mol/LNaOH： 10ml
Add aqua sterilisa to 100ml, abundant mixing, room temperature preservation, preferably matching while using.
The SolutionIII:100ml system:
KOAc： 29.4g
CH
3COOH： 11.5ml
Add aqua sterilisa to 100ml, 4 ℃ of preservations behind the autoclave sterilization.
Sequencing and analysis: sequencing is finished by Shanghai Ying Jun Bioisystech Co., Ltd.Institute's calling sequence in analyses such as Genebank and molecular biology software DNAMAN, ClustalW, is obtained SNP site information (Fig. 1).
(6) CAPS method
Adopt dCAPS 2.0 (http://helix.wustl.edu/dcaps/dcaps.html) software design primer, primer sequence is as follows:
CAPS1: forward primer 5 '-TGTTTGGAGAAAGTTGAAAGTC-3 '
Reverse primer 5 '-ACCATACAAACCTATCTCTATT-3 '
CAPS2: forward primer 5 '-GGAAGTGAGGTGTTGAATT-3 '
Reverse primer 5 '-TACACATTGGTCCGTTAGAC-3 '
CAPS3: forward primer 5 '-AGACGTAGCATTGCTTCTCTAG-3 '
Reverse primer is 5 '-TGGCATCCTTCAGCACCTTC-3 '
Reaction system and condition are the same, and the PCR product adopts AccI respectively, and EcoRI and XbaI restriction enzyme carry out enzyme to be cut, and three kinds of restriction endonucleases all adopt the reaction system of 20 μ l as follows:
Reaction system:
PCR product 5-10 μ l
0.1％BSA 2μl
10×buffer 2μl
Enzyme 5U
Replenish dd H
2O to 20 μ l;
Mixing solutions is placed 37 ℃, and the TaqI system places under 65 ℃ of conditions and respectively is incubated 4 hours, and product adopts HDA-GT12TM, and (eGene, USA) capillary electrophoresis system detects.
Utilize CAPS labeled analysis F2 population genetic rule, 3 SNP sites all are 1: 2: 1 segregation ratio (table 1), meet Mendelian's monogenic inheritance rule, so the CAPS method can effectively be distinguished resistance and responsive plant.
The SNP site that table 1, CAPS method are identified is at F
2Heredity separates from generation to generation
| The site | Primer | The FF type | The Ff type | The ff type | Virtual | Ratio |
| Site | ||||||
| 1 | CAPS1 | 23 | 47 | 25 | 95 | 1∶2∶1 |
| |
CAPS2 | 21 | 48 | 25 | 94 | 1∶2∶1 |
| |
CAPS3 | 23 | 46 | 23 | 92 | 1∶2∶1 |
(7) AS-PCR method
Place 3 ' of primer to hold in the SNP site, synthetic allele-specific primers, 2 and 3 places have designed 5 AS-PCR primers in the site for we, pairing obtains 6 combination of primers, optimize the PCR reaction conditions, it is right to screen an Auele Specific Primer, directly distinguishes high anti-and high sense germplasm by PCR method.
Primer is as follows to sequence:
AS-PCR: forward primer is 5 '-GTGTAACACAAATTCCTCAACA-3 '
Reverse primer is 5 '-GACGTAGCATTGCTTCTGTAGA-3 '.
(25 μ l) is as follows for the PCR reaction system:
dNTP(10mmol/L) 0.5μl
10 * PCR buffer (adds MgCl
2) (25mmol/L) 2.5 μ l
Each 0.5 μ l of upstream and downstream primer (10 μ mol/L)
Genomic dna 1.0 μ l
Distilled water is settled to 25 μ l
Reaction conditions: 94 ℃ of sex change 5min, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 45s, totally 30 circulations, 72 ℃ are extended 7min, adopt 3% agarose gel electrophoresis detection PCR product.
Choose 100 strain F immediately
2Separate plant for muskmelon heredity and carry out above-mentioned test, the purpose band clearly that increases of 76 strains as a result, 24 strains do not have band, and the single-gene that meets 3: 1 separates genetics rule (table 2), so the AS-PCR method can effectively be distinguished Fom-2 resistance and responsive plant.
The SNP site 2 and 3 that table 2, AS-PCR method are identified is at F
2Heredity separates from generation to generation
Choose more thin skin of Zhejiang area cultivated area and thick-skinned melon variety and local feature breed, amount to by 34 (table 3).Adopt AS-PCR and CAPS method that it is carried out the fusarium wilt disease resistance screening.
CAPS method wherein, we adopt specificity amplification primer: forward primer CAPS2-F:5 '-GGAAGTGAGG-TGTTGAATT-3 ' and reverse primer CAPS2-R:5 '-TACACATTGGTCCGTTAGAC-3 '), be that template is carried out specific PCR (reaction system and PCR reaction conditions are the same) with variety genome DNA to be measured.Adopt EcoR I restricted type restriction endonuclease to carry out enzyme behind the product purification and cut HAD-GT12
TMFull-automatic kapillary foranalysis of nucleic acids system carries out electrophoretic analysis, after data are proofreaied and correct, and record and analytical results.
In the AS-PCR method, we adopt specificity amplification primer: forward primer 2-3F1:5 '-GTGTAACACAAA-TTCCTCAACA-3 ' and reverse primer 2-3R1:5 '-GTGTAACACAAATTCCTCATCA-3 '), with variety genome DNA to be measured is that template is carried out specific PCR (reaction system and PCR reaction conditions are the same), product detects through 1.5% agarose gel electrophoresis, observe electrophoresis result, record and analytical results.
Table 3, common muskmelon Cultivar Fom-2 type and fusarium wilt disease resistance are identified
We adopt AS-PCR and two kinds of methods of CAPS that the conventional cultivation kind is carried out the Fom-2 functional molecular marker, and two kinds of methods obtain identical result.As can be seen from the test results, we screen 11 parts of fusarium wilt disease resistance materials from 34 portions of Cultivars, wherein comprise 2 in the disease-resistant variety of isozygotying, and 9 in heterozygosis disease-resistant variety is for fusarium wilt disease resistance breeding from now on provides good germ plasm resource.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO：1
(Fom-2-R)
gagtctattg?tttcgcttca?taatttgcaa?acattaaagt?tcctatactc?atttgttgaa 60
gaatttccaa?tgaacttttc?aaatttggta?aatttgaggc?acttgaaatt?atggcgaaat 120
gttaaccaaa?cgcctccaca?tttaagtcaa?ttgactcaac?ttcaaacatt?gtctcatttt 180
gtaatcgggt?ttgaagaagg?ttgtaagatt?attgaattgg?gaccattgaa?aaacttgcaa 240
ggtagtttga?atcttttgtg?tttggagaaa?gttgaaagta?aagaggaagc?caaaggagca 300
aacttggcag?aaaaggagaa?tttaaaagag?ctaaacttaa?gttggtccat?gaaaagaaaa 360
gataacgata?attacaatga?tttggaagtg?ttggaaggac?ttcaaccaaa?ccaaaatctc 420
caaatattaa?gaatccacga?ctttacagaa?aggcgtttgc?ctaacaagat?ttttgttgag 480
aatttaatag?agataggttt?gtatggttgt?gataattgtg?aaaagcttcc?aatgcttgga 540
caattaaaca?acttaaagaa?acttgagatt?tgcagctttg?atggcgttca?aattatagac 600
aacgagttct?atggtaatga?ttcaaaccaa?agaaggttct?tcccaaagct?tgagaaattt 660
gtaatgaatg?gtatgatgaa?cttagagcaa?tgggaagagg?taatgacaaa?tgatgcatca 720
tcaaatgtta?caatttttcc?caatcttata?agcttggaga?taagtggatg?tcccaaatta 780
acaaagattc?caaacggatt?acacttttgt?agttccattc?gacgtgtaaa?aatataccaa 840
tgttcaaatt?tgagcataaa?tataagaaat?aagccggaat?tatggtattt?acacattggt 900
ccgttagaca?agctaccaga?agatttatgt?catctcatga?atttgggggt?aatgacaatt 960
gttggaaata?tacagaatta?tgattttggc?atccttcagc?accttccttc?ccttaaaaaa 1020
attactttag?tcgaggatga?gttgagcaat?aatagtgtaa?cacaaattcc?tcaacaactt 1080
caacacctca?cttccctgga?atttctgtca?attgaaaatt?ttggaggcat?cgaagctttg 1140
ccagaatggc?taggaaactt?ggtatgtttg?caaacactct?gttttctttg?ttgcagaaat 1200
ttgaaaaaac?taccttctac?agaagcaatg?ctacgtctca?ctaaattaaa?taaattgtat 1260
gcttgcgaat?gtccaatgct?actactcgaa?gaaggtgacc?cagagcgagc?a 1311
SEQ?ID?NO：2
(Fom-2-S)
gagtctattg?tttcgcttca?taatttgcaa?acattaaagt?tcctatactc?atttgttgaa 60
gaatttccaa?tgaacttttc?aaatttggta?aatttgaggc?acttgaaatt?atggcgaaat 120
gttaaccaaa?cgcctccaca?tttaagtcaa?ttgactcaac?ttcaaacatt?gtctcatttt 180
gtaatcgggt?ttgaagaagg?ttgtaagatt?attgaattgg?gaccattgaa?aaacttgcaa 240
ggtagtttga?atcttttgtg?tttggagaaa?gttgaaagta?gagaggaagc?caaaggagca 300
aacttggcag?aaaaggagaa?tttaaaagag?ctaaacttaa?gttggtccat?gaaaagaaaa 360
gataacgata?attacaatga?tttggaagtg?ttggaaggac?ttcaaccaaa?ccaaaatctc 420
caaatattaa?gaatccacga?ctttacagaa?aggcgtttgc?ctaacaagat?ttttgttgag 480
aatttaatag?agataggttt?gtatggttgt?gataattgtg?aaaagcttcc?aatgcttgga 540
caattaaaca?acttaaagaa?acttgagatt?tgcagctttg?atggcgttca?aattatagac 600
aacgagttct?atggtaatga?ttcaaaccaa?agaaggttct?tcccaaagct?tgagaaattt 660
gtaatgaatg?gtatgatgaa?cttagagcaa?tgggaagagg?taatgacaaa?tgatgcatca 720
tcaaatgtta?caatttttcc?caatcttata?agcttggaga?taagtggatg?tcccaaatta 780
acaaagattc?caaacggatt?acacttttgt?agttccattc?gacgtgtaaa?aatataccaa 840
tgttcaaatt?tgagcataaa?tataagaaat?aagccggaat?tatggtattt?acacattggt 900
ccgttagaca?agctaccaga?agatttatgt?catctcatga?atttgggggt?aatgacaatt 960
gttggaaata?tacagaatta?tgattttggc?atccttcagc?accttccttc?ccttaaaaaa 1020
attactttag?tcgaggatga?gttgagcaat?aatagtgtaa?cacaaattcc?tcaacgactt 1080
caacacctca?cttccctgga?atttctgtca?attgaaaatt?ttggaggcat?cgaagctttg 1140
ccagaatggc?taggaaactt?ggtatgtttg?caaacactct?gttttctttg?ttgcagaaat 1200
ttgaaaaaac?tacctcctac?agaagcaatg?ctacgtctca?ctaaattaaa?taaattgtat 1260
gcttgcgaat?gtccaatgct?actactcgaa?gaaggtgacc?cagagcgagc?a 1311
SEQ?ID?NO：3
tgctcctttg?gcttcctgt 19
SEQ?ID?NO：4
gagtctattg?tttcgcttca 20
SEQ?ID?NO：5
ggaagtgagg?tgttgaatt 19
SEQ?ID?NO：6
tacacattgg?tccgttagac 20
SEQ?ID?NO：7
agacgtagca?ttgcttctct?ag 22
SEQ?ID?NO：8
tggcatcctt?cagcaccttc 20
SEQ?ID?NO：9
gtgtaacaca?aattcctcaa?ca 22
SEQ?ID?NO：10
gacgtagcat?tgcttctgta?ga 22
Claims (2)
1. one kind is used for the functional molecular marker that the muskmelon fusarium wilt disease resistance is identified, it is characterized in that:
The CAPS primer sequence that described molecule marker adopted be following any one:
CAPS1: forward primer is 5 '-TGCTCCTTTGGCTTCCTGT-3 '
Reverse primer is 5 '-GAGTCTATTGTTTCGCTTCA-3 ',
CAPS2: forward primer is 5 '-GGAAGTGAGGTGTTGAATT-3 '
Reverse primer is 5 '-TACACATTGGTCCGTTAGAC-3 ',
CAPS3: forward primer is 5 '-AGACGTAGCATTGCTTCTCTAG-3 '
Reverse primer is 5 '-TGGCATCCTTCAGCACCTTC-3 ';
Perhaps, the AS-PCR primer sequence that described molecule marker adopted is:
AS-PCR: forward primer is 5 '-GTGTAACACAAATTCCTCAACA-3 '
Reverse primer is 5 '-GACGTAGCATTGCTTCTGTAGA-3 '.
2. the purposes of functional molecular marker as claimed in claim 1 is characterized in that: be used for the marker assisted selection of muskmelon blight, perhaps be used to identify muskmelon fusarium wilt disease resistance gene.
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| CN 201110378256 Division CN102399887B (en) | 2010-04-15 | 2010-04-15 | Functional molecular marker 3 for fusarium wilt resistance identification and purpose thereof |
| CN 201110377951 Division CN102399885B (en) | 2010-04-15 | 2010-04-15 | Functional molecular marker I for identifying muskmelon fusarium wilt resistance and application thereof |
| CN 201110378228 Division CN102399886B (en) | 2010-04-15 | 2010-04-15 | Functional molecular marker 2 for fusarium wilt resistance identification and purpose thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140506A (en) * | 2010-12-16 | 2011-08-03 | 新疆农业科学院哈密瓜研究中心 | Molecular marker linked with gummy stem blight resistance gene Gsb-2 and application thereof |
| CN103243152A (en) * | 2012-02-11 | 2013-08-14 | 邵元健 | Bilateral molecular marker identification technology of melon wilt |
| CN103348242A (en) * | 2011-01-12 | 2013-10-09 | 积水医疗株式会社 | Method for detecting single nucleotide polymorphisms |
| CN103866005A (en) * | 2014-01-21 | 2014-06-18 | 东北农业大学 | Molecular marker-assisted selection primer and method for wilt resistance and gynoecious line of cucumis melon |
| CN103882017A (en) * | 2014-03-25 | 2014-06-25 | 北京市农林科学院 | Molecular marker of cucumber fusarium wilt anti-disease gene Foc-4 as well as special primer and application thereof |
| US9481881B2 (en) | 2011-01-12 | 2016-11-01 | Sekisui Medical Co., Ltd. | Eluent for ion-exchange chromatography, and method of analyzing nucleic acid chains |
| CN108277296A (en) * | 2018-04-17 | 2018-07-13 | 中国农业科学院植物保护研究所 | A kind of identification of the Fusarium strain for causing root rot and differentiating method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763226A (en) * | 2005-09-20 | 2006-04-26 | 杭州师范学院 | Method for identifying strong female cucumber variety Mantian 705 by CAPS marker |
-
2010
- 2010-04-15 CN CN2010101476500A patent/CN101899437B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763226A (en) * | 2005-09-20 | 2006-04-26 | 杭州师范学院 | Method for identifying strong female cucumber variety Mantian 705 by CAPS marker |
Non-Patent Citations (3)
| Title |
|---|
| 《The Plant Journal》 20040831 Tarek Joobeur，et al. The fusarium wilt resistance locus Fom-2 of melon contains a single resistance gene with complex features 283-297页 1-2 第39卷, 第3期 2 * |
| 《生物技术》 20060630 李金玉 等 甜瓜抗枯萎病基因同源序列克隆与序列分析 3-9页 1-2 第16卷, 第3期 2 * |
| 《第12次全国西瓜甜瓜科研生产协作会议学术交流论文摘要集》 20090424 王士伟 等 基于SNP的甜瓜枯萎病抗性基因Fom-2功能性分子标记的开发 19 1-2 , 2 * |
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| CN102140506A (en) * | 2010-12-16 | 2011-08-03 | 新疆农业科学院哈密瓜研究中心 | Molecular marker linked with gummy stem blight resistance gene Gsb-2 and application thereof |
| CN103348242A (en) * | 2011-01-12 | 2013-10-09 | 积水医疗株式会社 | Method for detecting single nucleotide polymorphisms |
| CN103348242B (en) * | 2011-01-12 | 2016-08-17 | 积水医疗株式会社 | The detection method of single nucleotide polymorphism |
| US9447460B2 (en) | 2011-01-12 | 2016-09-20 | Sekisui Medical Co., Ltd. | Method for detecting single nucleotide polymorphisms |
| US9481881B2 (en) | 2011-01-12 | 2016-11-01 | Sekisui Medical Co., Ltd. | Eluent for ion-exchange chromatography, and method of analyzing nucleic acid chains |
| CN103243152A (en) * | 2012-02-11 | 2013-08-14 | 邵元健 | Bilateral molecular marker identification technology of melon wilt |
| CN103866005A (en) * | 2014-01-21 | 2014-06-18 | 东北农业大学 | Molecular marker-assisted selection primer and method for wilt resistance and gynoecious line of cucumis melon |
| CN103866005B (en) * | 2014-01-21 | 2015-08-19 | 东北农业大学 | The primer of Muskmelon Fusarium wilt resistance and complete female system molecular marker assisted selection and method |
| CN103882017A (en) * | 2014-03-25 | 2014-06-25 | 北京市农林科学院 | Molecular marker of cucumber fusarium wilt anti-disease gene Foc-4 as well as special primer and application thereof |
| CN108277296A (en) * | 2018-04-17 | 2018-07-13 | 中国农业科学院植物保护研究所 | A kind of identification of the Fusarium strain for causing root rot and differentiating method |
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