CN102399887B

CN102399887B - Functional molecular marker 3 for fusarium wilt resistance identification and purpose thereof

Info

Publication number: CN102399887B
Application number: CN 201110378256
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: 2013-08-07
Anticipated expiration: 2030-04-15
Also published as: CN102399887A

Abstract

The invention discloses a functional molecular marker 3 for fusarium wilt resistance identification, which adopts a primer sequence of CAPS3. A forward primer is 5'-AGACGTAGCATTGCTTCTCTAG-3', and a reverse primer is 5'-TGGCATCCTTCAGCACCTTC-3'. The functional molecular marker 3 is used for fusarium wilt molecular auxiliary breeding or is used for fusarium wilt resistance gene identification.

Description

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

The present invention is to be 2010.04.15 the applying date, and application number is dividing an application of 201010147650.0 " being used for functional molecular marker that the muskmelon fusarium wilt disease resistance identifies 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 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 the 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 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-rich repeat, 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 identifies the nontoxic gene that produces variation, and this is conducive to people and selects in molecular level discovery and the knowledge of natural environment.Now people from many species the clone obtain the R gene, and verified that they are at 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 be conducive to 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 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 functional molecular marker relevant with 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 for the evaluation of muskmelon fusarium wilt disease resistance,

The CAPS primer sequence that this molecule marker adopts 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 adopts of this molecule marker 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 for the identification of 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:

CAPS1: 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 muskmelon idioplasm to the genotype (table 3) of 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 zone SNP 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 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 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 database, with the disease-resistant and susceptible germplasm of muskmelon and their F ₁, F ₂From generation to generation genomic dna is 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 reliability.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 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, the 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, grind under the liquid nitrogen state, add the 2 * CTAB (30 μ l mercaptoethanol mixing) of 65 ℃ of preheatings of 600 μ l, transfer in the centrifuge tube, 65 ℃ the insulation 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 acetic acid ammonia

75% (volume ratio) ethanol

Reagent and the condition of (4) chain polymerization enzyme reaction (PCR):

At first following reagent is mixed in together

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 ₄DNALigase 10×Buffer： 1μl

PUCM-T carrier: 1 μ l

PCR product: 4 μ l

PEG4000： 1μl

T ₄DNALigase： 1μl；

(b) preparation (CaCl of competent cell ₂Legal system is equipped with competent cell):

1. the single bacterium colony of the E.coli DH5 α of the new activation of picking 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 with aseptic rifle choicest, be inoculated in the 5mlLB nutrient solution that contains 50 μ g/ml Amp 37 ℃ of overnight incubation.

(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 Solution II, 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.

Solution III: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:

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 F ₂The 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 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:

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 the 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 '), be that template is carried out specific PCR (reaction system and PCR reaction conditions are the same) with variety genome DNA to be measured, 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.

Claims

1. be used for the functional molecular marker three that the muskmelon fusarium wilt disease resistance is identified, it is characterized in that:

The primer sequence that described molecule marker three adopts is:

CAPS3: forward primer is 5 '-AGACGTAGCATTGCTTCTCTAG-3 '

Reverse primer is 5 '-TGGCATCCTTCAGCACCTTC-3 '.

2. the purposes of functional molecular marker three as claimed in claim 1 is characterized in that: adopt above-mentioned label screening to identify muskmelon anti-blight germplasm, be used for the marker assisted selection of muskmelon blight.