CN101899439B - Specific fragment closely associated with resistance gene Pm-2F of melon powdery mildew and obtainment method thereof - Google Patents
Specific fragment closely associated with resistance gene Pm-2F of melon powdery mildew and obtainment method thereof Download PDFInfo
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Abstract
The invention discloses a specific fragment closely associated with a resistance gene Pm-2F of melon powdery mildew, and belongs to the field of biotechnology. The specific fragment is shown as sequence tables SEQ ID NO:2 and SEQ ID NO:4. The obtained SSR specific fragment has the advantages of good repeatability, stable and reliable markers, convenient statistics and the like; and a specific amplification sequence of the specific fragment is the basis of developing other specific markers, and has high utilization value on the identification of resistant and susceptible varieties.
Description
The application is dividing an application of following patent application:
Application number: 200710178022.7
The applying date: on November 23rd, 2007
Denomination of invention: with the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F and preparation method thereof
Technical field
The present invention relates to and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F and preparation method thereof, belong to biological technical field.
Background technology
Muskmelon is the annual herbaceous plant that overgrows of Curcurbitaceae Cucumis (Cucumis melo L.), is a kind of Important Economic crop of extensively cultivating both at home and abroad.The muskmelon cultivation history in existing more than 3000 year of China is one of country of cultivating the earliest in the world muskmelon, also is the country that the muskmelon cultivated area is maximum in the world at present, output is the highest.
Powdery Mildew is a kind of worldwide disease that the harm muskmelon is produced, and in many countries and regions generation is arranged, and has a strong impact on the yield and quality of muskmelon, even can cause total crop failure, has caused the extensive attention of countries in the world.Powdery Mildew is mainly caused by melon list softgel shell bacterium (Sphaerotheca fuliginea) and two spore powdery mildews (Erysiphe cichoracearum).At present people more in depth are studied pathogenic, the route of transmission of pathogenic bacteria, preventive measures etc., also do a lot of work for seeking relevant anti-source gene, but the harm of Powdery Mildew still has the trend of development, and the seed selection of carrying out disease-resistant variety is the effective way that addresses this problem.Therefore, the research of cucurbits powdery mildew breeding for disease resistance become one of major objective of breeding work.
The traditional breeding method mode wastes time and energy, and needs very large colony that the resistance of Powdery Mildew is strictly screened.Utilize with the closely linked specific mark fragment of disease-resistant proterties and carry out screening varieties, can greatly shorten the research cycle of traditional breeding method, accelerate the transfer of target gene.At present, be mainly derived from various molecular marking techniques with the acquisition of the closely linked specific mark fragment of disease-resistant proterties, such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellite DNA or simple repeated sequence (SSR), distinguished sequence amplification (SCAR), single nucleotide polymorphism (SNP) etc.Wherein, RAPD and the detection of AFLP labeling technique are subject to the impact of envrionment conditions and cause the experimental result poor reproducibility, SCAR and SNP labeling technique need to utilize known dna sequence design special primer to increase, and the SSR labeling technique has good reproducibility, mark is reliable and stable, being convenient to the advantages such as statistics, is marking tool more stable in the present practical application, and the specific fragment sequence of utilizing the SSR labeling technique to obtain also is the important foundation of other molecule markers such as exploitation SCAR and SNP.Therefore, this research intends utilizing the SSR labeling technique to seek and the closely linked specific mark fragment of melon powdery mildew resistance gene, this will be the basis of carrying out from now on the melon powdery mildew resistance molecular breeding, and thus obtained extension increasing sequence is the basis of the specific molecular markers such as exploitation SCAR and SNP.
Summary of the invention
The object of the present invention is to provide a kind of and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, this specific fragment can be used in the powdery mildew of melon molecular mark.
Another object of the present invention provides the preparation method of this specific fragment.
For achieving the above object, the present invention adopts following technical scheme:
A kind of and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, it has SEQ ID NO:1 in the sequence table～4 described dna sequence dnas.
SEQ ID NO:1 is the sequence of disease-resistant specific amplified fragment SSR509-172;
SEQ ID NO:2 is the sequence of disease-resistant specific amplified fragment SSR510-98 in the sequence table;
SEQ ID NO:3 is the sequence of susceptible specific amplified fragment SSR509-170 in the sequence table;
SEQ ID NO:4 is the sequence of susceptible specific amplified fragment SSR510-104 in the sequence table.
A kind of preparation method of and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, described method comprises the steps:
(1) with the screening of the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F: extract genomic dna, use the SSR primer that genomic dna is carried out the PCR reaction, the PCR product is carried out electrophoretic analysis; By the BSA method, seek and the closely linked special SSR fragment of melon powdery mildew resistance gene Pm-2F, measure linkage distance and verify linkage relationship;
(2) with the acquisition of the closely linked specific amplified sequence of melon powdery mildew resistance gene Pm-2F: to the SSR product reclaim, Cloning and sequencing, obtain the specific fragment sequence.
The SSR primer is SSR primer 509 and SSR primer 510 in the preparation method of the above-mentioned a kind of and closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, described step (1);
Described SSR primer 509 upstream primer sequences are: 5 '-CTGGCCCCCTCCTAAACTAA-3 ', 509 downstream primer sequences are 5 '-CAAAAAGCATCAAAATGGTTG-3 '.
Described SSR primer 510 upstream primer sequences are: 5 '-TTTCACTTTTTCCCGCCG-3 ', 510 downstream primer sequences are 5 '-AATGGAAAAGGGAAGTGCAA-3 '.
The preparation method of the above-mentioned a kind of and closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, wherein, extraction genomic dna in the described step (1) refers to 1.5 gram blades grind into powder in liquid nitrogen is added 9ml 2%CTAB extracting solution, 65 ℃ of water-baths of mixing 1 hour; From water-bath, take out centrifuge tube, add 1/3 volume 5M Potassium ethanoate, mixing ice bath 20 minutes; Add equal-volume chloroform/primary isoamyl alcohol extracting twice; Get supernatant and add 2/3 volume isopropanol precipitating DNA; The lavation buffer solution washing once dries up, and adds the dissolving of TE damping fluid; Add RNase A and make its final concentration reach 100 μ g/ml, 37 ℃ of water-baths of mixing 1 hour; With equal-volume chloroform/primary isoamyl alcohol extracting; Get supernatant, add 1/2 volume 7.5M ammonium acetate and 2 times of volume dehydrated alcohol precipitation DNA; 70% washing with alcohol precipitation dries up, and adds an amount of ddH
2The O dissolving DNA.
The preparation method of the above-mentioned a kind of and closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, wherein, the PCR reaction conditions in the described step (1) is: contain 2.5 μ l, 10 * buffer in the reaction system of 25 μ l, 2.0 μ l 25mM MgCl
2, 2.0 μ l 2.5mM dNTP, 1U TaqDNA polysaccharase, 30ng SSR primer, 50ng template DNA; Response procedures is, 94 ℃ of denaturation 5min, and 94 ℃ of 1min then, 52 ℃ of 1min, circulation is 35 times under 72 ℃ of 2min, and 72 ℃ are kept under 4 ℃ of conditions after extending 7min.
The preparation method of the above-mentioned a kind of and closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, wherein, recovery, the Cloning and sequencing of the SSR product in the described step (2) refers to prove chain SSR specific fragment with the recovery of Gene-Clean test kit purifying, then be connected on the pGEM-T Vector Easy carrier, recombinant plasmid dna is carried out enzyme with EcoRI cut processing, verify amplified production with SSR-PCR, whether the fragment that observation amplifies is consistent with original purpose fragment and endonuclease bamhi, and order-checking.
The present invention uses simple repeated sequence (Simple Sequence Repeat, SSR) technology, at muskmelon RIL (Recombinant Inbred Line, RIL) adopt Bulk segregant analysis (Bulked Segregating Analysis in the colony, BSA) screened the closely linked SSR specific fragment with melon powdery mildew resistance gene Pm-2F, and SSR specific fragment cloning and sequencing has been obtained the specific amplified sequence.
Advantage of the present invention is: the SSR specific fragment that the present invention obtains has good reproducibility, and mark is reliable and stable, is convenient to the advantages such as statistics, and its specific amplified sequence also is the basis of other specific mark of exploitation.
The present invention will be further described below in conjunction with accompanying drawing and preferred forms, so that the public has whole to summary of the invention and understand fully, and is not restriction to protection domain of the present invention.Aforementioned part fully discloses the protection domain that the present invention can implement, and therefore allly any well known in the artly is equal to replacement according to what the disclosure of invention was carried out, all belongs to infringement of the present invention.
Description of drawings
Fig. 1 is that parents, F are felt in primer SSR509 and 510 antagonism
1, anti-sense pond DNA cloning figure as a result;
Fig. 2 is primer SSR509 to the pcr amplification of recombinant inbred lines figure as a result;
Fig. 3 is primer SSR510 to the pcr amplification of recombinant inbred lines figure as a result;
Fig. 4 is primer SSR509 to the pcr amplification of Germplasm Resources of Cucumis Melo L figure as a result;
Fig. 5 is primer SSR510 to the pcr amplification of Germplasm Resources of Cucumis Melo L figure as a result.
Embodiment
The acquisition of embodiment and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F and extension increasing sequence thereof
One, materials and methods:
1.1 material
Supplying examination material parent is Japanese type muskmelon material K7-1 and typical Xinjiang honey melon strain K7-2.Two materials provide by the Wu of academy of agricultural sciences, Xinjiang jewel academician.Male parent K7-1 is Japanese high mildew-resistance material, and the high sense of maternal K7-2 Powdery Mildew is typical Xinjiang honey melon, and the fruit pol is high, and meat is crisp, and shelf-lives is long, and special aromatising flavour is arranged.Hybridize take the two as the parent, from their filial generation F
2Stochastic sampling selfing in the material passes acquisition F by simple grain
8RIL consists of RIL colony, and totally 106 strains are used for specific fragment and sequence research.
Mark for further checking obtains has also adopted the distinctive 120 parts of good Germplasm Resources of Cucumis Melo Ls in this laboratory, 13 parts of disease-resistant germ plasm resources wherein, 107 parts of susceptible germ plasm resources.
1.2 research method
1.2.1 the extraction of genomic dna
Method (Murray M with reference to (1980) such as Murry, Thompson W F.Rapid isolation of high molecular weight plant DNA[J] .Nucl Acid Res, 1980,8:668-673.) carry out DNA extraction after being improved.
With 1.5 gram blades grind into powder in liquid nitrogen, add 9ml 2%CTAB extracting solution (2%CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0,20mM EDTA pH8.0,1%PVP-40,0.2% beta-mercaptoethanol), 65 ℃ of water-baths of mixing are 1 hour; From water-bath, take out centrifuge tube, add 1/3 volume 5M Potassium ethanoate, mixing, ice bath 20 minutes; Add twice of equal-volume chloroform/primary isoamyl alcohol (24: 1) extracting; Get supernatant and add 2/3 volume isopropanol precipitating DNA; Lavation buffer solution (76% ethanol, 10mM ammonium acetate) washing once dries up, and adds TE damping fluid (10mM Tri s-HCl, 1mM EDTA, pH7.4) dissolving; Add RNase A and make its final concentration reach 100 μ g/ml, 37 ℃ of water-baths of mixing 1 hour; With equal-volume chloroform/primary isoamyl alcohol (24: 1) extracting; Get supernatant, add 1/2 volume 7.5M ammonium acetate and 2 times of volume dehydrated alcohol precipitation DNA; 70% washing with alcohol precipitation dries up, and adds an amount of ddH
2The O dissolving DNA.
DNA concentration uses ultraviolet spectrophotometer (Shimadzu UV-1201, Japan) with the OD260 pH-value determination pH, and detects the DNA extraction quality with 0.8% agarose gel electrophoresis.
1.2.2PCR the detection of amplification and product
Contain 2.5 μ l, 10 * buffer in the reaction system of 25 μ l, 2.0 μ l 25mM MgCl
2, 2.0 μ l 2.5mM dNTP, 1U Taq archaeal dna polymerase, 30ng primer, 50ng template DNA.Taq archaeal dna polymerase and reaction buffer are available from TaKaRa company.DNTP is available from Shanghai Sangon company.The SSR primer is synthetic by Beijing AudioCodes biotech firm.
Response procedures is, 94 ℃ of denaturation 5min, and 94 ℃ of 1min then, 52 ℃ of 1min, circulation is 35 times under 72 ℃ of 2min, and 72 ℃ are kept under 4 ℃ of conditions after extending 7min.The PCR instrument is the PTC-100 that U.S. Biorad company makes.
Get each 5 μ l of amplified production and load sample damping fluid (98% methane amide, 10mM EDTA, 0.25% bromjophenol blue, 0.25% dimethylbenzene green grass or young crops) and mix, 94 ℃ of sex change 5min place cooled on ice immediately.Each sample is got 5 μ l loadings, adopts 6% polyacrylamide gel, the permanent power electrophoretic separation of 75W, and 2/3 place that goes to glue to the blue or green indicator of dimethylbenzene finishes electrophoresis.Silver dyes and manifests result and analysis.
1.2.3 the linkage relationship of specific amplified PCR product and powdery mildew resistance gene
Silver is analyzed electrophoretic band after dying and manifesting pcr amplification product.The banding pattern that isozygotys from male parent K7-1 is designated as " a ", is designated as " b " from the banding pattern that isozygotys of maternal K7-2, and band smudgy or that lose is designated as "-".Utilize the linkage relationship of Joinmap3.0 software analysis specific amplified PCR product and powdery mildew resistance gene.
1.2.4 the acquisition of specific amplified sequence
After determining that there are the close linkage relation in pcr amplification product and powdery mildew resistance gene, use ddH
2O rinses offset plate well, cut differential band with blade, be immersed in (20% ethanol in the high-salt buffer, 1M LiCl, 10mM Tris) 24h, then chloroform extracting dries up after the ethanol precipitation, be dissolved among the ddH2O, get 5 μ l as template again amplification under the PCR condition identical with selective amplification.Product detects at 1.5% agarose, if be defined as object tape, adopts the gel purification test kit Gene-Clean of Bo Ao biotech firm that the purpose fragment is carried out purifying.PCR product and carrier be connected the PGEM-T easy Vector system that adopts Promega company.Carry out conversion and the screening of recombinant plasmid with reference to the method in " molecular cloning experiment guide ".Utilize day little extraction reagent kit of the plasmid of root biotech firm to extract recombinant plasmid, adopt enzyme blanking method and PCR method that it is detected.To send through the plasmid that PCR and enzyme are cut the test positive clone in Beijing AudioCodes biotech firm and check order and the result is provided.
1.2.5 male parent, female parent, F
1With RIL disease-resistant inoculated identification in seedling stage
1.2.5.1 Powdery Mildew source and the source preparation of inoculation bacterium
Collect the Powdery Mildew on the pumpkin from national vegetables Engineering Technical Research Centre folium ilicis chinensis farm, adopt the spore suspension spray method to be inoculated on the Pumpkin Seedlings after the purifying breeding, be used for the inoculated identification of Powdery Mildew.
Get the beaker that spore on the sick leaf blade of summer squash places sterilized water, the preparation spore suspension that stirs, blood counting chamber counting conidium number with writing brush type brush.Inoculum density is 2.0 * 10
5Individual spore/mL.
1.2.5.2 seedling stage disease-resistant inoculated identification
Male parent, female parent, F
1With RIL F
8106 strains, respectively get 20 seeds, establish 2 repetitions, repeat 10 young plants 1 time.Seed binds up with gauze, and after 55 ℃ of sterilizations of hoting water treatment of seeds, soaks 4h again, places 28 ℃ of constant incubator vernalization 18 hours, is sowed in the 72 holes dish that bactericidal nurishing soil is housed, grow in the air-conditioning greenhouse, the daytime/night temperature be 25～28 ℃/18～20 ℃.After cotyledon flattens, adopt spore powder spray method inoculation S.fuliginea microspecies 2F, inoculate 12 to 15 days and institute an inquiry anti-sense reaction after abundant the morbidity.
1.2.5.3 seedling stage disease-resistant inoculated identification grade scale
Be divided into 6 grades.0 grade: whole strain is without any scab; 1 grade: only cotyledon has and seldom measures scab; 2 grades: only cotyledon has more scab or cotyledon that scab is arranged, and has on the stem and seldom measures scab; 3 grades: cotyledon has a lot of scabs, and a small amount of scab is arranged on the stem; 4 grades: be covered with scab on cotyledon and the stem; 5 grades: plant is dead.
1.2.5.4 male parent, female parent, F
1With phenotypic the settling the standard of RIL anti-sense in seedling stage
Determine male parent, female parent, F according to disease index DI
1Anti-sense phenotype with RIL.
Disease index DI=(∑ rank * strain number) * 100/ (total strain number * 6).
Disease index is decided to be susceptible greater than 40, be decided to be disease-resistant less than 40.
Two, results and analysis
2.1 melon powdery mildew resistance gene Pm-2F Inheritance Analysis on Genetic
Enantiopathy parent K7-1, Susceptible parent K7-2, F
1, RIL F
8106 strains carry out disease-resistant inoculated identification in seedling stage, according to the sickness rate of each strain of Powdery Mildew severity Scaling standard survey and calculate disease index.The result shows, K7-1, K7-2 and F
1Disease index be respectively 0.00,93.03 and 3.44, show as respectively disease-resistantly, susceptible, disease-resistant, illustrate that muskmelon K7-1 is to be controlled by dominant gene to the resistance of Powdery Mildew S.fuliginea physiological strain 2F.And the disease-resistant qualification result of RIL shows that 106 strains have 58 disease-resistant strains and 48 susceptible strains, separates than the theoretical ratio that meets 1: 1 card square check χ
2=0.94＜α
2 0.05,1=3.84, show that muskmelon K7-1 is one pair of genes control to the resistance of Powdery Mildew S.fuliginea physiological strain 2F.Comprehensive parents, F
1With the disease-resistant qualification result in seedling stage of 106 strains of RIL, muskmelon K7-1 is controlled by pair of dominant genes Pm-2F to the resistance of Powdery Mildew S.fuliginea physiological strain 2F.
2.2 with the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F
Adopt group's hybrid analysis method to obtain and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F.
According to RIL disease-resistant qualification result in seedling stage, respectively get 5 parts of anti-sense strain DNA that isozygoty and mix the anti-sense gene pool of structure.With disease-resistant parent K7-1, Susceptible parent K7-2, F
1With anti-sense gene pool be template, 660 pairs of SSR combination of primers are carried out pcr amplification, filter out the combination of primers that can between parents and anti-sense gene pool, produce the notable difference band.660 pairs of SSR primer sequences come from the paper (Danin-Poleg-2001 that has delivered at present, Genzalo et al.2005, Joobeur T et al, 2004, Fazio et al.2002) with according to muskmelon EST information storage (http://melon.bti.cornell.edu) designed, designed in the Internet.
The hybrid analysis result of group shows, only have the anti-sense parent amplified band of primer SSR509 and SSR510 consistent with the amplification bands of a spectrum in anti-sense pond in 660 pairs of SSR primers, as shown in Figure 1, Fig. 1 is the pcr amplification result that parent, F1, anti-sense pond are felt in primer SSR509 and SSR510 antagonism.(M.30-330bpmarker; 1,6.K7-2; 2,7.K7-1; 3,8.F1; 4,9. disease-resistant gene pond; 5,10. susceptible gene pool)
Described SSR primer 509 upstream primer sequences are: 5 '-CTGGCCCCCTCCTAAACTAA-3 ', 509 downstream primer sequences are 5 '-CAAAAAGCATCAAAATGGTTG-3 '.
Described SSR primer 510 upstream primer sequences are: 5 '-TTTCACTTTTTCCCGCCG-3 ', 510 downstream primer sequences are 5 '-AATGGAAAAGGGAAGTGCAA-3 '.
Utilizing primer SSR509 and SSR510 to carry out disease-resistant specific amplified fragment that pcr amplification obtains disease-resistant parent K7-1 names respectively and is SSR509-172 and SSR510-98.Utilizing primer SSR509 and SSR510 to carry out susceptible specific amplified fragment that pcr amplification obtains Susceptible parent K7-2 names respectively and is SSR509-170 and SSR510-104.
Utilize primer SSR509 and SSR510 respectively the strain of 106 RILs to be carried out pcr amplification.
In the pcr amplification of primer SSR509, as shown in Figure 2, Fig. 2 is primer SSR509 to the pcr amplification of recombinant inbred lines figure (1:K7-1 as a result; 2:K7-2; The 3-57:RTL strain).There are 56 strains only to amplify the disease-resistant specific fragment of SSR509-172 in 58 disease-resistant strains, 2 disease-resistant strains amplify the disease-resistant specific fragment of SSR509-172 and the susceptible specific fragment of SSR509-170 simultaneously, F7 and two seedling stages in generation of F8 disease-resistant inoculated identification result show that these 2 disease-resistant strains are the heterozygosis strain, so the pcr amplification result is consistent with the disease-resistant qualification result in field.Have 47 strains only to amplify the susceptible specific fragment of SSR509-170 in 48 susceptible strains, 1 strain amplifies the disease-resistant specific fragment of SSR509-172, namely only has 1 exchange strain in 106 RIL strains.Through the Joinmap3.0 software analysis, the linkage distance of disease-resistant specific fragment SSR509-172 and melon powdery mildew resistance gene Pm-2F is 1cm, presents the close linkage relation.
In the primer SSR510 amplification, as shown in Figure 3, Fig. 3 primer SSR510 is to the pcr amplification of recombinant inbred lines figure (1:K7-1 as a result; 2:K7-2; The 3-50:RIL strain; ).Have 55 strains only to amplify the disease-resistant specific fragment of SSR510-98 in 58 disease-resistant strains, 2 disease-resistant strains amplify the disease-resistant specific fragment of SSR510-98 and the susceptible specific fragment of SSR510-104, F simultaneously
7And F
8Two seedling stages in generation, disease-resistant inoculated identification result showed that these 2 disease-resistant strains were the heterozygosis strain, and 1 disease-resistant strain only amplifies the susceptible specific fragment of SSR510-104, showed that this disease-resistant strain is the exchange strain.Have 47 strains only to amplify the susceptible specific fragment of SSR510-104 in 48 susceptible strains, 1 susceptible strain amplifies the disease-resistant specific fragment of SSR510-98, illustrates that this susceptible strain also is the exchange strain.Therefore, have 2 exchange strains in 106 RIL strains.Through the Joinmap3.0 software analysis, the linkage distance of disease-resistant specific fragment SSR510-98 and melon powdery mildew resistance gene Pm-2F is 3cM, presents the close linkage relation.
For further confirming the linkage relationship of disease-resistant specific fragment SSR509-172, SSR510-98 and melon powdery mildew resistance gene Pm-2F, utilize 120 parts of good Germplasm Resources of Cucumis Melo L materials in this laboratory to verify.The disease-resistant qualification result in field shows in 120 parts of Germplasm Resources of Cucumis Melo L materials that 13 disease-resistant varieties and 107 susceptible variety are arranged.
The pcr amplification result of SSR509 shows, as shown in Figure 4, Fig. 4 is the pcr amplification figure as a result of 509 pairs of Germplasm Resources of Cucumis Melo Ls of primer SSR; Have 4 kinds to amplify the disease-resistant specific fragment of SSR509-172 in 13 disease-resistant varieties, the bands of a spectrum that all the other kinds amplify are in the another location; Have 101 in 107 susceptible variety and amplify the susceptible specific fragment of SSR509-170, with the disease-resistant qualification result in the field rate of coincideing be 87.5%.
The SSR510 amplification shows, as shown in Figure 5, Fig. 5 is the pcr amplification figure as a result of 510 pairs of Germplasm Resources of Cucumis Melo Ls of primer SSR; There are 4 kinds to amplify the disease-resistant specific fragment of SSR510-98 in 13 disease-resistant varieties, have 83 to amplify the susceptible specific fragment of SSR510-104 in 107 susceptible variety, reach 72.5% with field investigation result's identical rate.
Above-mentioned pcr amplification result has further confirmed the close linkage relation of disease-resistant specific fragment SSR509-172 and SSR510-98, susceptible specific fragment SSR509-170 and SSR510-104 and melon powdery mildew resistance gene Pm-2F, accuracy is high, illustrates that above-mentioned labeled fragment has very high utility value when differentiating the resistant, susceptible kind.
2.4 the sequencing result of specific fragment
Disease-resistant specific amplified fragment SSR509-172 and SSR510-98 and susceptible specific amplified fragment SSR509-170 and SSR510-104 are checked order.There is respectively the upstream and downstream binding site of primer SSR509 and SSR510 at the two ends of 4 order-checking fragments, and the size of fragment with estimate according to molecular weight standard substantially identical, confirmed the exactness of order-checking fragment.Anti-, the sense specific fragment length of utilizing 509 amplifications of SSR primer to obtain are respectively 172bp and 170bp, only have the insertion of 2 bases " CT ".Anti-, the sense specific fragment length of utilizing 510 amplifications of SSR primer to obtain are respectively 98bp and 102bp, only have the disappearance of 4 bases " CTCT ".
Claims (2)
1. one kind and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F is characterized in that it is shown in sequence table SEQ ID No.2 and SEQ ID No.4.
2. the preparation method with the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F is characterized in that described method comprises the steps:
(1) with the screening of the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F: extract genomic dna, use the SSR primer that genomic dna is carried out the PCR reaction, the PCR product is carried out electrophoretic analysis; By the BSA method, seek and the closely linked special SSR fragment of melon powdery mildew resistance gene Pm-2F, measure linkage distance and verify linkage relationship;
(2) with the acquisition of the closely linked specific amplified sequence of melon powdery mildew resistance gene Pm-2F: to the SSR product reclaim, Cloning and sequencing, obtain the specific fragment sequence;
The SSR primer is SSR primer 510 in the described step (1);
Described SSR primer 510 upstream primer sequences are: 5 '-TTTCACTTTTTCCCGCCG-3 ', and 510 downstream primer sequences are 5 '-AATGGAAAAGGGAAGTGCAA-3 ';
Described and the closely linked specific fragment of melon powdery mildew resistance gene Pm-2F, its sequence is shown in sequence table SEQ ID No.2 and SEQ ID No.4;
Extraction genomic dna in the described step (1) refers to 1.5 gram blades grind into powder in liquid nitrogen is added the 9ml2%CTAB extracting solution, 65 ℃ of water-baths of mixing 1 hour; From water-bath, take out centrifuge tube, add 1/3 volume 5M Potassium ethanoate, mixing ice bath 20 minutes; Add equal-volume chloroform/primary isoamyl alcohol extracting twice; Get supernatant and add 2/3 volume isopropanol precipitating DNA; The lavation buffer solution washing once dries up, and adds the dissolving of TE damping fluid; Add RNase A and make its final concentration reach 100 μ g/ml, 37 ℃ of water-baths of mixing 1 hour; With equal-volume chloroform/primary isoamyl alcohol extracting; Get supernatant, add 1/2 volume 7.5M ammonium acetate and 2 times of volume dehydrated alcohol precipitation DNA; 70% washing with alcohol precipitation dries up, and adds an amount of ddH2O dissolving DNA;
PCR reaction conditions in the described step (1) is: contain 2.5 μ l10 * buffer in the reaction system of 25 μ l, 2.0 μ l25mM MgCl
2, 2.0 μ l2.5mM dNTP, 1U Taq archaeal dna polymerase, 30ng SSR primer, 50ng template DNA; Response procedures is, 94 ℃ of denaturation 5min, and 94 ℃ of 1min then, 52 ℃ of 1min, circulation is 35 times under 72 ℃ of 2min, and 72 ℃ are kept under 4 ℃ of conditions after extending 7min;
Recovery, the Cloning and sequencing of the SSR product in the described step (2) refers to prove chain SSR specific fragment with the recovery of Gene-Clean test kit purifying, then be connected on the pGEM-TVector Easy carrier, recombinant plasmid dna is carried out enzyme with EcoRI cut processing, verify amplified production with SSR-PCR, whether the fragment that observation amplifies is consistent with original purpose fragment and endonuclease bamhi, and order-checking.
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| CN107164481B (en) * | 2017-06-01 | 2022-12-13 | 中国农业科学院蔬菜花卉研究所 | Powdery mildew resistance related SSR (simple sequence repeat) marker for muskmelon and application of powdery mildew resistance related SSR marker |
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