CN101899437B

CN101899437B - Functional molecular marker for resistance identification of blight in Cucumis melo L. and application thereof

Info

Publication number: CN101899437B
Application number: CN2010101476500A
Authority: CN
Inventors: 杨景华; 张明方; 王士伟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2010-04-15
Filing date: 2010-04-15
Publication date: 2012-04-25
Anticipated expiration: 2030-04-15
Also published as: CN101899437A

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

Be used for functional molecular marker of muskmelon fusarium wilt disease resistance evaluation and uses thereof

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 a kind of novel molecular mark is provided for the marker assisted selection of muskmelon fusarium wilt disease resistance.

Background technology

Muskmelon (Cocumis melo L.) has higher commerce article 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 (the 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) through the method for map based cloning.The Fom-2 gene belongs to the R gene of NBS-LRR class, and (nucleotide binding site is NBS) with LRR (leucine-richrepeat, LRR) two structural domains of relatively guarding for this genoid coding NBS.It is the identification owing to the nontoxic gene of host's antagonism albumen generation that common plant specially changes resistance, and then causes 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.Many researchs show that there are the polymorphum of sequence in Arabidopis thaliana and other several species R genes, 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 easy generation reorganization exchanges and separates, thus the accuracy of influence selection.

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 need 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 one type of novel molecular mark, can improve the efficient of mark greatly.

Summary of the invention

The technical problem that the present invention will solve 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 present 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 property 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 nucleotide sequence such as the sequence table SEQ ID NO:1 of muskmelon fusarium wilt disease resistance gene Fom-2 are said.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 such as sequence table SEQ ID NO:2 are said.

SNP characteristic 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

Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.

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

Embodiment 1, key step of the present invention and content are: according to muskmelon fusarium wilt disease resistance gene Fom-2 design primer in the DB, with the disease-resistant and susceptible germplasm of muskmelon and their F ₁, F ₂From generation to generation genomic dna is a template, chain polymerization enzyme reaction (PCR) method amplification purpose fragment, order-checking, and the applying biological information science is analyzed sequence between right then.Adopt two kinds of relevant FMs of method exploitation fusarium wilt disease resistance of CAPS and AS-PCR, and at F ₁, F ₂Verify from generation to generation its validity and safety.Use above-mentioned two kinds of marking methods, extract to produce and go up the different muskmelon idioplasm genomic dnas of using, identify the genotype of its fusarium wilt disease resistance.

Concrete steps are following:

(1) in ncbi database (http://www.ncbi.nlm.nih.gov/), find the sequence information of muskmelon fusarium wilt disease resistance gene Fom-2, 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, the design primer at two ends, Fom-2 gene LRR district, primer sequence is following:

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 deposition, 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 subsequent use.

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

Template DNA 1 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

TV 50 μ l

The PCR reaction conditions is: behind 94 ℃ of preparatory 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 following:

(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 subsequent use.

(c) heat shock method transformed into escherichia coli DH5 α:

1. get the competent cell suspension-s 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 with the rifle head gently, 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:

Each component below adding 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 cultures 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 adds isopyknic phenol/chloroform mixing;

(g) 4 ℃, centrifugal 5 minutes of 12000rpm, supernatant 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 deposition, 50 μ l TE melt deposition, and-20 ℃ of preservations are subsequent use.

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 existing with join at present.

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 accomplished 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 following:

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 following:

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 value	Ratio
							Site 1	CAPS1	23	47	25	95	1∶2∶1
Site 2	CAPS2	21	48	25	94	1∶2∶1
							Site 3	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 through PCR method.

Primer is following to sequence:

AS-PCR: forward primer is 5 '-GTGTAACACAAATTCCTCAACA-3 '

Reverse primer is 5 '-GACGTAGCATTGCTTCTGTAGA-3 '.

PCR reaction system (25 μ l) is as follows:

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

Embodiment 2,

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, observes 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.Can find out that from 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, 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

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 AS-PCR primer sequence that said 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.