CN102140506B - Molecular marker linked with gummy stem blight resistance gene Gsb-2 and application thereof - Google Patents
Molecular marker linked with gummy stem blight resistance gene Gsb-2 and application thereof Download PDFInfo
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Abstract
The invention discloses a method for obtaining a molecular marker linked with gummy stem blight resistance gene Gsb-2 and application of the molecular marker. An F2 group is constructed by PI 157082 carrying the gummy stem blight resistance gene Gsb-2 and a local susceptible variety of white skin crisp, gummy stem blight conidiospore suspension is artificially inoculated in a seedling period, and the molecular marker ISSR-57560 linked with the Gsb-2 is obtained through a simple sequence repeat (ISSR) technology. The molecular marker ISSR-57560 is used for detecting genotypes of melon varieties or strains taking PI 157082 as a parent, and can judge that whether the varieties or strains resist gummy stem blight. The invention overcomes the defects of high difficulty and longer period in breeding stable gummy stem blight resistant strains in the prior art, and provides a marker-assisted selection technology for gummy stem blight resistant breeding so as to reduce cost, improve selection efficiency, and quicken the progress of culturing good stem blight resistant melon strains.
Description
Invention field
The present invention relates to agricultural biological technical field.Specifically, the present invention relates to the chain molecule marker of a kind of and Gummy Stem Blight Resistance in Melon gene Gsb-2 and the technical field of preparation method and application thereof.
Background technology
Muskmelon is a kind of important cash crop, and cultivated area and the output of China's muskmelon all rank first in the world.The gummy stem blight of melon that is caused by didymella bryoniae is one of serious global fungal disease.Its generation often can cause destructive consequence, and except the harm muskmelon, didymella bryoniae also infects other ground family crops such as cucumber, watermelon, pumpkin and summer squash, causes the underproduction in various degree.Not only efficient is low for the climing rot of chemical prevention, also easily causes environmental pollution, is to prevent and treat the safest, effective of gummy stem blight of melon harm and save one of measure of cost and cultivate disease-resistant variety.
In the prior art report, the efficient of the climing rot of chemical prevention is low, easily causes the problems such as pathogenic bacteria resistance and environmental pollution, and breeding resistant variety is the most long-term, the effective measures of control gummy stem blight of melon.The traditional method of breeding resistant variety is subject to artificially reach the impact of environmental factors, is difficult to filter out accurately and rapidly the individuality with disease-resistant gene, and molecular marker assisted selection then can overcome the limitation of traditional breeding way, improves efficiency of selection.Up to now, the gummy stem blight of melon resistance is mainly by 4 dominant controls of single-gene and 1 the recessive control of single-gene.The known anti-source of gummy stem blight of melon comprises PI 140471 (carrying resistant gene Gsb-1), PI 157082 (Gsb-2), PI 511890 (Gsb-3), PI 482398 (Gsb-4) and PI 482399 (gsb-5) at present, about the research of these 5 resistant gene molecule markers there is not yet report (FRANTZ and JAHN, 2004).
In order to filter out accurately and rapidly the individual plant with disease-resistant gene.Prior art is to adopt artificial inoculation on seedling to identify during by traditional method seed selection breeding for disease resistance material, screen according to the anti-sense feature of the phenotype of plant, traditional method because inoculation is insufficient or onset condition is not suitable for and affect efficiency of selection, is difficult to filter out accurately and rapidly the individual plant with disease-resistant gene sometimes.The molecular mark technology that grew up in recent years, traditional Phenotypic Selection can be changed into direct Select gene type by molecular marker assisted selection,, all have great importance from reducing the aspects such as cost, raising selection for just can selecting by the enantiopathy plant early.
Summary of the invention
The objective of the invention is for the deficiencies in the prior art, a kind of molecule marker and the application thereof chain with Gummy Stem Blight Resistance in Melon gene Gsb-2 are provided.Overcome prior art and stablized the defective that the disease-resistant strain difficulty of gummy stem blight of melon is large, the cycle is long in seed selection, the technology of marker assisted selection is provided for the climing rot breeding for disease resistance of muskmelon, thereby reduce cost, improve efficiency of selection, accelerate to cultivate the process of good anti didymella muskmelon strain.
The present invention is achieved through the following technical solutions: by making up the anti-sense of gummy stem blight of melon F
2For colony, find the molecule marker chain with Gummy Stem Blight Resistance in Melon gene Gsb-2, reach seed selection anti didymella strain process and shorten breeding cycle, improve and select the good technique effect of accuracy rate.
The present invention specifically provides a kind of molecule marker chain with Gummy Stem Blight Resistance in Melon gene Gsb-2, may further comprise the steps:
(1) take muskmelon resource PI 157082 as the anti-source of climing rot, the climing rot resistant gene Gsb-2 that selects PI 157082 to carry, resistant gene Gsb-2 is dominant inheritance.
(2) the susceptible local variety of gummy stem blight of melon " white skin is crisp " are hybridized for male parent with the disease-resistant resource PI 157082 of gummy stem blight of melon for maternal, obtain hybrid F
1
(3) by hybrid F
1Self-pollination obtains F
2For colony, extract each F
2Genomic dna for colony's individual plant.
(4) by the Disease Resistance Identification in seedling stage, inoculation melon didymella bryoniae conidial suspension 14d " Invest, Then Investigate " disease situation, different according to muskmelon stem Lesion size, disease is divided into 1-5 grade, and according to the disease scale situation, be the disease-resistant or susceptible phenotype of single plant with the classification number conversion.
(5) from F
2In colony, the genomic dna of getting the disease-resistant a plurality of individual plant equivalent of phenotype makes up the disease-resistant gene mixing pit, and the genomic dna of a plurality of individual plant equivalent that phenotype is susceptible makes up susceptible gene mixing pit.
(6) take disease-resistant gene pond and susceptible gene pool DNA as template, carry out the screening amplification of polymorphism ISSR primer, the ISSR amplified production obtains polymorphism ISSR primer after electrophoretic separation on 1.5% sepharose.
(7) between disease-resistant gene pond and susceptible gene pool, have polymorphism 3 primer I SSR-12, ISSR-45 and an ISSR-57 according to acquired, further with disease-resistant resource PI 157082, susceptible local variety " white skin is crisp ", hybrid F
1And the disease-resistant F that is used for making up anti-sense gene pool
2Individual plant, susceptible F
2The individual plant checking obtains the molecule marker ISSR-57 chain with the gummy stem blight of melon resistant gene
560
(8) according to acquired molecule marker ISSR-57
560, to F
2For colony's individual plant augmentation detection and carry out genetic analysis, obtain chain molecule marker data.
(9) with F
2Disease-resistant, susceptible phenotype and molecule marker for colony's individual plant carry out the genetic linkage analysis, take 3.0 as minimum LOD threshold values, determine the molecule marker ISSR-57 chain with gummy stem blight of melon resistant gene Gsb-2
560
Among the present invention, between disease-resistant gene pond and susceptible gene pool, have among polymorphism 3 primer I SSR-12, ISSR-45 and the ISSR-57 according to acquired, described polymorphic molecular marker, its primer sequence is:
ISSR-12 5′GAGGAGGAGGAGGAGGAG′3；
ISSR-45 5′ACACACACACACACACGC′3；
ISSR-57 5′AGAGAGAGAGAGAGAGTG′3。
Among the present invention, investigate in the disease situation behind the inoculation melon didymella bryoniae, different according to muskmelon stem Lesion size, disease is divided into 1-5 grade, concrete grade scale is as follows: 1 grade=without scab; 2 grades=single scab (1-10mm) or several continuous scab (1-20mm) but not around one week of stem; 3 grades=several continuous scabs (21-80mm) or scab are around one week of stem; The dehydration atrophy of 4 grades=stem but plant are dead; 5 grades=plant is dead, because genetic analysis needs, 1 grade of disease and 2 grades is decided to be disease-resistant, and 4 grades and 5 grades of diseases are decided to be susceptible.
Among the present invention, with 134 strain F
2Disease-resistant, susceptible phenotype and the molecule marker of individual plant carry out the genetic linkage analysis, take 3.0 as minimum LOD threshold values, determine the molecule marker ISSR-57 chain with gummy stem blight of melon resistant gene Gsb-2
560
Molecule marker and the application thereof chain with gummy stem blight of melon resistant gene Gsb-2 involved in the present invention are with molecule marker ISSR-57
560The genotype detection that is used for melon variety or strain is used for judging whether anti-gummy stem blight of melon of this kind or strain, may further comprise the steps:
(1) with disease-resistant resource PI 157082 with other muskmelons hybridization and multiply to F
2More than generation;
(2) the single plant of muskmelon to obtaining by step (1) is extracted genomic dna, whether has the molecule marker mutually chain with climing rot resistant gene Gsb-2 among the genomic DNA of Detection and Extraction; If any resistance molecule marker ISSR-57
560Occur, whether the prediction Muskmelon Plants anti didymella.
Selected anti-source PI 157082 materials among the present invention derive from USDA country plant germplasm resource system (USDA NPGS), and those of ordinary skills can obtain by the introduction approach.
Selected muskmelon local variety among the present invention " white skin is crisp " derive from Xinjiang Agricultural Sciences institute hami melon research centre, and those of ordinary skills can obtain by buying or granting approach.
Selected F among the present invention
1, deriving from disease-resistant material PI 157082 (male parent), those of ordinary skills can obtain by the conventional hybridization approach.
Selected F among the present invention
2, derive from F
1Selfing produces, and those of ordinary skills can obtain by conventional selfing approach.
The present invention is male parent with PI 157082 and derived varieties thereof or product or maternally hybridizes with other muskmelon and multiply to F
2More than generation.Described PI 157082 and derived varieties or strain, refer to take PI 157082 as the parent, by conventional hybridization or adopt tissue culture or adopt anther culture or with other physics or chemical process hybridized induction monoploid, double melon variety or the strain that reagent doubles to obtain double haploid or adopts the genetic transforming method acquisition with colchicine and other plant chromosome again, these technique means are well known to those of ordinary skill in the art.
The sequence information of the ISSR primer of the present invention's screening can freely obtain by general biological website, biotech company's reagent catalogue or the paper of having published.
The molecule marker ISSR-57 chain with Gummy Stem Blight Resistance in Melon gene Gsb-2 provided by the present invention
560In, with the DNA of the anti-source PI 157082 of primer I SSR-57 amplification gummy stem blight of melon, can amplify length is the labeled fragment band of 560bp, shows and carries resistant gene Gsb-2.
By implementing the concrete summary of the invention of the present invention, can reach following effect:
1. obtain first the ISSR molecule marker chain with Gummy Stem Blight Resistance in Melon gene Gsb-2 by the present invention.
2. clear and definite by molecule marker of the present invention location anti didymella gene Gsb-2, it is convenient to identify.By detecting the molecule marker chain with anti didymella gene Gsb-2, can predict the climing rot resistance of Muskmelon Plants, rapid screening disease-resistant variety or strain are used for the muskmelon breeding.Detect disease-resistant gene Gsb-2 fast and easy, and not affected by environment.
3. by molecule marker provided by the invention, the assistant breeding select target is clear and definite, saves cost.In traditional breeding way, at first to collect parent with disease-resistant gene and Cultivar and carry out a series of hybridization and backcross and set up colony, and will carry out individual plant to climing rot resistance in the colony and select.Gummy stem blight of melon is carried out artificial inoculation on seedling identify, because inoculating insufficient or onset condition is not suitable for affecting efficiency of selection and accuracy rate.Therefore breeding for disease resistance is not only time-consuming, and difficulty is large, and cost is high.By mark ISSR-57
560Detect anti didymella gene Gsb-2, can just identify in seedling stage the individual plant of anti didymella, eliminate other plant, not only save production cost but also greatly improve the efficiency of selection of anti didymella muskmelon.
Description of drawings
Fig. 1 is shown as primer I SSR-57 to parents, F
1, make up the disease-resistant individual plant of anti-sense gene pool and the augmentation detection of susceptible individual plant.
Fig. 2 is shown as primer I SSR-57 to F
2The augmentation detection of individual plant.
Among Fig. 1-2: M be Ladder DNA, 1 for PI 157082,2 crisp for white skin, 3 for F
1, R is that disease-resistant individual plant, S are susceptible individual plant.
Embodiment
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.
Equipment and material have among the present invention: TC-512 type pcr amplification instrument (Britain Techne company), JY3000 type electrophoresis apparatus (Beijing monarch anticipate east electrophoresis equipment company limited), JY-SPE type electrophoresis chamber (Beijing monarch anticipate east electrophoresis equipment company limited), the digital gel imaging instrument of JY04S-3C type (Beijing monarch anticipate east electrophoresis equipment company limited), dATP among ISSR primer, Taq enzyme, the dNTPs, dCTP, dGTP, dTTP respectively get 10mM composition mixed solution and all are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Other reagent is purchased from Haikou dawn chemical industry Instr Ltd..
The breeding material that relates among the present invention comprises: Gummy Stem Blight Resistance in Melon resource PI 157082, research and analyse PI 157082 through genetic development and carry climing rot resistant gene Gsb-2, resistant gene Gsb-2 shows as dominant inheritance, and anti-source PI 157082 draws the plant germplasm resource system from USDA country.Muskmelon local variety " white skin is crisp " are for originating in farmers''s conventional variety in Xinjiang, the climing rot of susceptible.
Among the present invention, 1.5% sepharose adopts in the 100ml TAE solution and contains the 1.5g agarose.
All reagent of selecting among the present invention and instrument all are well known selecting, but do not limit enforcement of the present invention.
Embodiment one: the ISSR molecule marker ISSR-57 chain with Gummy Stem Blight Resistance in Melon gene Gsb-2
560Acquisition
1) disease-resistant material PI 157082 (male parent) and Xinjiang Melon local variety " white skin is crisp " (female parent) are hybridized acquisition F
1, F
1Selfing produces F
2By plantation parents, F
1Generation and F
2For colony.To its incidence that carries out artificial inoculation and add up each individual plant, F
2Show that for the segregating population statistics in the 134 group of hill bodies, 83 strains performance is disease-resistant, 36 strains performance is susceptible, meets the hereditary pattern of 3: 1 single dominant gene.
2) the climing rot of inoculation and disease scale: adopt the method (Zhang et al., 1997) of conidial suspension spray inoculation and 5 grades of disease scales, spray spore suspension with micro-sprayer, spore suspension concentration is 5 * 105 mL
-1, till being sprayed onto plant leaf and beginning to drip.With little shed moisturizing, relative humidity is opened little shed more than 90% behind the 3d after the inoculation, 2 all " Invest, Then Investigate " statistics state of an illness.Carry out classification and the anti-sense phenotype of recording individual according to the base of the plant disease.1 grade=without scab; 2 grades=single scab (1-10mm) or several continuous scab (1-20mm) but not around one week of stem; 3 grades=several continuous scabs (21-80mm) or scab are around one week of stem; The dehydration atrophy of 4 grades=stem but plant are dead; 5 grades=plant is dead.The disease rank is that 1 or 2 individual record is disease-resistant, and the disease rank is that 4 or 5 individual record is susceptible.
3) structure in the extraction of genomic dna and DNA pond: extracting genome DNA adopts the CTAB method.Utilize the colour developing of agarose gel electrophoresis and ethidium bromide to carry out the quantitative analysis of DNA concentration.After the F2 individual plant DNA extraction, utilize the BSA method to get the DNA balanced mix of 5 disease-resistant individual plants and 5 susceptible individual plants, be built into the polymorphism screening that disease-resistant and susceptible DNA pond is used for the ISSR primer.The primer sequence that is used for the screening polymorphism sees Table 1.
Table 1:ISSR primer numbering and sequence
4) the ISSR-PCR reaction system is: 10 * buffer (does not contain Mg
2+) 2.0 μ L; DNTP (2mmolL
-1) 2.0 μ L; MgCl
2(25mmolL
-1) 1.5 μ L; Primer (10mmolL
-1) 1.0 μ L; Template DNA (30ng μ L
-1) 1.0 μ L; Taq polysaccharase (5U μ L
-1) 0.2 μ L; DdH
2O 12.3 μ L, totally 20 μ L.Pcr amplification carries out at TC-512 amplification instrument (TECHNE), and response procedures is as follows: 94 ℃ of denaturation 5min, then 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 35 circulations, at 72 ℃ of lower extension 10min, 4 ℃ of preservations, about 2.5h of whole response procedures of continuing.
5) agarose electrophoresis detects: get 10 μ LPCR reaction product and mix with 1.5 μ L tetrabromophenol sulfonphthaleins (0.25%), point sample is in containing 0.5 μ gmL
-1In 1.5% sepharose of EB, take 1 * TAE as electrophoretic buffer, at 5Vcm
-1Under the stabilized voltage about electrophoresis 1.5h.Take a picture with the digital gel imaging instrument of JY04S-3C.
6) linksystem analysis: utilize Mapmaker (Version 3.0) software that F2 is carried out genetic linkage analysis for mark and the anti-type representation of data of segregating population individual plant, and utilize the Kosambi function that recombination fraction is converted into genetic distance (cM), adopt Mapmaker (Version 3.0) software that the ISSR mark of acquisition and the genetic affinity between the gene Gsb-2 are carried out the linksystem analysis, the result shows: there are linkage relationship in this mark and resistant gene Gsb-2, its genetic linkage distance is 11.3cM, names to be ISSR-57
560。
Embodiment two: primer I SSR-57 is to parents' augmentation detection
As template, carry out the ISSR-PCR amplification with primer I SSR-57, the ISSR-PCR reaction system is: 10 * buffer (does not contain Mg take the crisp genomic dna of parents PI 157082 and Bai Pi
2+) 2.0 μ L; DNTP (2mmolL
-1) 2.0 μ L; MgCl
2(25mmolL
-1) 1.5 μ L; Primer (10mmolL
-1) 1.0 μ L; Template DNA (30ng μ L
-1) 1.0 μ L; Taq polysaccharase (5U μ L
-1) 0.2 μ L; DdH
2O 12.3 μ L, totally 20 μ L.The pcr amplification reaction program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s then, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 35 circulations, continue to extend 10min, 4 ℃ of preservations, the about 2.5h of whole response procedures down at 72 ℃.
Amplified production mixes with 1.5 μ L tetrabromophenol sulfonphthaleins (0.25%), and point sample is in containing 0.5tgmL
-1In 1.5% sepharose of EB, take 1 * TAE as electrophoretic buffer, at 5Vcm
-1Under the stabilized voltage about electrophoresis 1.5h.Detect with the digital gel imaging instrument of JY04S-3C, anti-source PI 157082 can amplify the band of a treaty 560bp, and local variety " white skin is crisp " then do not amplify band at same position.
Embodiment three: primer I SSR-57 is to F
1Augmentation detection
With hybrid F
1Genomic dna be template, carry out ISSR-PCR amplification and electrophoresis detection with primer I SSR-57, amplification and detection method are: the ISSR-PCR reaction system is: 10 * buffer (does not contain Mg
2+) 2.0 μ L; DNTP (2mmolL
-1) 2.0 μ L; MgCl
2(25mmolL
-1) 1.5 μ L; Primer (10mmolL
-1) 1.0 μ L; Template DNA (30ng μ L
-1) 1.0 μ L; Taq polysaccharase (5U μ L
-1) 0.2 μ L; DdH
2O 12.3 μ L, totally 20 μ L.The pcr amplification reaction program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s then, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 35 circulations, continue to extend 10min, 4 ℃ of preservations down at 72 ℃.Amplified production mixes with 1.5 μ L tetrabromophenol sulfonphthaleins (0.25%), and point sample is in containing 0.5 μ gmL
-1In 1.5% sepharose of EB, take 1 * TAE as electrophoretic buffer, at 5Vcm
-1About electrophoresis 1.5h, detect with the digital gel imaging instrument of JY04S-3C under the stabilized voltage.F
1Can amplify the band of a treaty 560bp.
Embodiment four: primer I SSR-57 is to F
2Augmentation detection
With 134 strain F
2The genomic dna of each individual plant of colony is template, carries out ISSR-PCR amplification and electrophoresis detection with primer I SSR-57, and amplification and detection method are: the ISSR-PCR reaction system is: 10 * buffer (does not contain Mg
2+) 2.0 μ L; DNTP (2mmolL
-1) 2.0 μ L; MgCl
2(25mmolL
-1) 1.5 μ L; Primer (10mmolL
-1) 1.0 μ L; Template DNA (30ngtL
-1) 1.0 μ L; Taq polysaccharase (5U μ L
-1) 0.2 μ L; DdH
2O 12.3 μ L, totally 20 μ L.The pcr amplification reaction program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s then, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 35 circulations, continue to extend 10min, 4 ℃ of preservations down at 72 ℃.Amplified production mixes with 1.5 μ L tetrabromophenol sulfonphthaleins (0.25%), and point sample is in containing 0.5 μ gmL
-1In 1.5% sepharose of EB, take 1 * TAE as electrophoretic buffer, at 5Vcm
-1About electrophoresis 1.5h, detect with the digital gel imaging instrument of JY04S-3C under the stabilized voltage.F
277 individual plants can amplify the band of a treaty 560bp in the colony, and 41 individual plants do not amplify band at same position.
Embodiment five: primer I SSR-57 is to 5 disease-resistant F
2The augmentation detection of individual plant
To make up 5 disease-resistant F of disease-resistant gene mixing pit
2The genomic dna of individual plant is template, carries out ISSR-PCR amplification and electrophoresis detection with primer I SSR-57, and amplification and detection method are: the ISSR-PCR reaction system is: 10 * buffer (does not contain Mg
2+) 2.0 μ L; DNTP (2mmolL
-1) 2.0 μ L; MgCl
2(25mmolL
-1) 1.5 μ L; Primer (10mmolL
-1) 1.0 μ L; Template DNA (30ng μ L
-1) 1.0 μ L; Taq polysaccharase (5U μ L
-1) 0.2 μ L; DdH
2O 12.3 μ L, totally 20 μ L.The pcr amplification reaction program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s then, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 35 circulations, continue to extend 10min, 4 ℃ of preservations down at 72 ℃.Amplified production mixes with 1.5 μ L tetrabromophenol sulfonphthaleins (0.25%), and point sample is in containing 0.5 μ gmL
-1In 1.5% sepharose of EB, take 1 * TAE as electrophoretic buffer, at 5Vcm
-1About electrophoresis 1.5h, detect with the digital gel imaging instrument of JY04S-3C under the stabilized voltage.5 individual plants all can amplify the band of a treaty 560bp.
Embodiment six: primer I SSR-57 is to 5 susceptible F
2The augmentation detection of individual plant
To make up 5 susceptible F of susceptible gene mixing pit
2The genomic dna of individual plant is template, carries out ISSR-PCR amplification and electrophoresis detection with primer I SSR-57, and amplification and detection method are: the ISSR-PCR reaction system is: 10 * buffer (does not contain Mg
2+) 2.0 μ L; DNTP (2mmolL
-1) 2.0 μ L; MgCl
2(25mmolL
-1) 1.5 μ L; Primer (10mmolL
-1) 1.0 μ L; Template DNA (30ng μ L
-1) 1.0 μ L; Taq polysaccharase (5U μ L-1) 0.2 μ L; DdH
2O 12.3 μ L, totally 20 μ L.The pcr amplification reaction program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s then, 55 ℃ of annealing 45s, 72 ℃ are extended 2min, carry out 35 circulations, continue to extend 10min, 4 ℃ of preservations down at 72 ℃.Amplified production mixes with 1.5 μ L tetrabromophenol sulfonphthaleins (0.25%), and point sample is in containing 0.5 μ gmL
-1In 1.5% sepharose of EB, take 1 * TAE as electrophoretic buffer, at 5Vcm
-1About electrophoresis 1.5h, detect with the digital gel imaging instrument of JY04S-3C under the stabilized voltage.5 individual plants all do not amplify band at same position.
Embodiment seven:
F take muskmelon resource PI 157082 as the parent
2, F
3Progeny population after planting, get plant cotyledon or blade and extract genomic dna, with primer I SSR-57 DNA is carried out ISSR-PCR amplification and electrophoresis detection, amplification and detection method are the same, carry the disease-resistant plant of anti didymella gene Gsb-2 performance and can amplify about 560bp band, and the plant of the climing rot of the climing rot gene of nonreactive Gsb-2 performance sense can not amplify band at same position.From extracting genomic dna to obtaining qualification result, in testing laboratory, only need to finish 1-2 working days.
Identify whether to carry anti didymella gene Gsb-2 by traditional method, then need muskmelon to be identified hybridized first with PI 140471 (carrying resistant gene Gsb-1), PI 157082 (Gsb-2), PI 511890 (Gsb-3), PI 482398 (Gsb-4) and PI 482399 (gsb-5) respectively and obtain F
1, again selfing and backcross and make up F
2And BC
1Colony in conjunction with the artificial inoculation melon didymella bryoniae, just can obtain qualification result according to the phenotypic inheritance of anti-sense.Each hybridization, selfing and the combination that backcrosses are calculated according to 200 individual plants, need at least to plant the 1000 group of hill body plant pedestrian worker that goes forward side by side and inoculate didymella bryoniae.According to 100 days seasons of growth calculating of muskmelon, traditional method just can identify and whether carry anti didymella gene Gsb-2 after identifying and needing at least 200 days.
Anti didymella gene Gsb-2 is clear and definite by molecule marker of the present invention location, and it is convenient to identify.By detecting the molecule marker chain with anti didymella gene Gsb-2, can predict the climing rot resistance of Muskmelon Plants, rapid screening disease-resistant variety or strain are used for the muskmelon breeding.Detect disease-resistant gene Gsb-2 fast and easy, and not affected by environment.
Embodiment eight:
F take muskmelon resource PI 157082 as the parent
2, F
3Progeny population after planting, get plant cotyledon or blade and extract genomic dna, with primer I SSR-57 DNA is carried out ISSR-PCR amplification and electrophoresis detection, utilize the disease-resistant individuality of molecular marker assisted selection, the plant of performance anti didymella can amplify about 560bp band, show plant not disease-resistant or that feel climing rot and then can not amplify band at same position, in testing laboratory, only need 1-2 working days and namely select disease-resistant individual plant.
Be parent's F by traditional method at muskmelon resource PI 157082
2, F
3Select the individuality of anti didymella in the progeny population, behind colony's insemination and emergence, artificial inoculation didymella bryoniae when plant to be planted is the 3rd leaf period, plant begins to show climing rot symptom behind the inoculation 14d, selects disease-resistant individual plant according to the anti-sense phenotype of plant.The optimal temperature of didymella bryoniae conidia germination is that 25-28 ℃, optimum relative humidity are more than 90%, onset condition requires stricter, the facilities environment that needs the subsidiary heat and moisture preserving support equipment of certain space, in addition, spray that conidial suspension is inhomogeneous, onset condition is unstable during inoculation, water deficit in a plant fertilizer deficiency etc. all causes Select Error easily.
In traditional breeding way, at first to collect parent with disease-resistant gene and Cultivar and carry out a series of hybridization and backcross and set up colony, and will carry out individual plant to climing rot resistance in the colony and select.Gummy stem blight of melon is carried out artificial inoculation on seedling identify, because inoculating insufficient or onset condition is not suitable for affecting efficiency of selection and accuracy rate.Therefore breeding for disease resistance is not only time-consuming, and difficulty is large, and cost is high.By mark ISSR-57
560Detect anti didymella gene Gsb-2, can just identify in seedling stage the individual plant of anti didymella, eliminate other plant, not only save production cost but also greatly improve the efficiency of selection of anti didymella muskmelon.
Claims (2)
1. molecule marker preparation method chain with Gummy Stem Blight Resistance in Melon gene Gsb-2 is characterized in that described preparation method may further comprise the steps:
(1) take muskmelon resource PI 157082 as the anti-source of climing rot, the climing rot resistant gene Gsb-2 that selects PI 157082 to carry, resistant gene Gsb-2 is dominant inheritance;
(2) the susceptible local variety of gummy stem blight of melon " white skin is crisp " for maternal and the disease-resistant resource PI157082 of gummy stem blight of melon are that male parent is hybridized, are obtained hybrid F
1
(3) by hybrid F
1Self-pollination obtains F
2For colony, extract each F
2Genomic dna for colony's individual plant;
(4) by the Disease Resistance Identification in seedling stage, inoculation melon didymella bryoniae conidial suspension 14d " Invest, Then Investigate " disease situation, different according to muskmelon stem Lesion size, disease is divided into 1-5 grade, and according to the disease scale situation, be the disease-resistant or susceptible phenotype of single plant with the classification number conversion;
(5) from F
2In colony, the genomic dna of getting the disease-resistant a plurality of individual plant equivalent of phenotype makes up the disease-resistant gene mixing pit, and the genomic dna of a plurality of individual plant equivalent that phenotype is susceptible makes up susceptible gene mixing pit;
(6) take disease-resistant gene pond and susceptible gene pool DNA as template, carry out the screening amplification of polymorphism ISSR primer, the ISSR amplified production obtains polymorphism ISSR primer after electrophoretic separation on 1.5% sepharose;
(7) between disease-resistant gene pond and susceptible gene pool, have polymorphism 3 primer I SSR-12, ISSR-45 and an ISSR-57 according to acquired, further with disease-resistant resource PI 157082, susceptible local variety " white skin is crisp ", hybrid F
1And the disease-resistant F that is used for making up anti-sense gene pool
2Individual plant, susceptible F
2The individual plant checking obtains the molecule marker ISSR-57 chain with the gummy stem blight of melon resistant gene
560
(8) according to acquired molecule marker ISSR-57
560, to F
2For colony's individual plant augmentation detection and carry out genetic analysis, obtain chain molecule marker data;
(9) with F
2Disease-resistant, susceptible phenotype and molecule marker for colony's individual plant carry out the genetic linkage analysis, take 3.0 as minimum LOD threshold values, determine the molecule marker ISSR-57 chain with gummy stem blight of melon resistant gene Gsb-2
560
In the described polymorphic molecular marker, polymorphism ISSR primer primer sequence is respectively:
ISSR-12 5′GAGGAGGAGGAGGAGGAG′3；
ISSR-45 5′ACACACACACACACACGC′3；
ISSR-57 5′AGAGAGAGAGAGAGAGTG′3。
2. a molecule marker application chain with Gummy Stem Blight Resistance in Melon gene Gsb-2 as claimed in claim 1 is characterized in that described molecule marker ISSR-57 that will be chain with the gummy stem blight of melon resistant gene
560Be used for the application of the genotype detection of melon variety or strain.
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| CN108754006B (en) * | 2018-05-31 | 2021-05-07 | 河南农业大学 | Molecular marker closely linked with muskmelon vine stem length character gene SI/SI |
| CN109735650B (en) * | 2019-02-11 | 2021-04-27 | 浙江大学 | Four single nucleotide polymorphism-based molecular markers for resisting gummy stem blight of melon and application thereof |
| CN109797238B (en) * | 2019-02-11 | 2021-06-01 | 浙江大学 | Two molecular markers developed based on gummy stem blight resistance identification and application thereof |
| CN110066884B (en) * | 2019-04-30 | 2023-10-20 | 黑龙江省科学院自然与生态研究所 | Clustered form-work ISSR-PCR molecular marker assisted breeding method |
| CN111057785B (en) * | 2020-01-17 | 2022-08-02 | 北京市农林科学院 | Molecular marker related to gummy stem blight resistance and application thereof |
| CN113881801B (en) * | 2021-10-29 | 2023-08-11 | 东北农业大学 | Molecular marker composition closely linked with deep green stem traits of cucurbita moschata and application of molecular marker composition |
| CN115125320A (en) * | 2022-06-01 | 2022-09-30 | 华中农业大学 | InDel marker co-separated from gummy stem blight resistance gene Gsb-3 and application thereof |
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| CN101440366B (en) * | 2007-11-23 | 2010-10-27 | 北京市农林科学院 | Specific fragment tightly linked to musk melon powdery mildew resistance gene Pm-2F and obtaining method thereof |
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