CN103555857A - Gene segment in close linkage with cucumber anti- and infected-alternaria leaf spot genes and resistance identification method of gene segment - Google Patents
Gene segment in close linkage with cucumber anti- and infected-alternaria leaf spot genes and resistance identification method of gene segment Download PDFInfo
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- CN103555857A CN103555857A CN201310576852.0A CN201310576852A CN103555857A CN 103555857 A CN103555857 A CN 103555857A CN 201310576852 A CN201310576852 A CN 201310576852A CN 103555857 A CN103555857 A CN 103555857A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a gene segment in close linkage with cucumber anti- and infected-alternaria leaf spot genes and a resistance identification method of the gene segment. The resistant identification method is used for identifying a primer sequence for identifying the cucumber alternaria leaf spot resistance. The primer consists of an upstream primer and a downstream primer, wherein the upstream primer has a sequence shown as SEQ ID No.1; the downstream primer has a sequence shown as SEQ ID No.2. The constructed F2-generation segregation population and cucumber genetic resources are subjected to alternaria leaf spot resistance evaluation by utilizing the primer sequence as well as cucumber anti-alternaria leaf spot gene segments and cucumber infected alternaria leaf spot gene segments amplified from the primer sequence, the result and artificial inoculation identification coincidence rate are respectively 95.60 percent and 93.42 percent, and the gene segment has a high application value for performing resource resistance screening during cucumber breeding. The sequences are taken as the basis, so that anti-alternaria leaf spot marked assistant selection and breeding can be performed, and the primer serves as a probe for detecting the cucumber anti-alternaria leaf spot genes during cucumber breeding. The anti-alternaria leaf spot gene localizing, plotting and cloning can be performed.
Description
Technical field
The present invention relates to technical field of biological genetic engineering, relate to method and the technology of cucumber conventional cross-breeding, molecular mark.Particularly with the closely linked gene fragment of the anti-black spot gene of cucumber, with the closely linked gene fragment of cucumber sense black spot gene, identify the method for resistance of cucumber against alternaria cucumerina.
Background technology
Cucumber (Cucumis Sativus L.) is 1 year herbaceous plant that overgrows of Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis), is the generally vegetables crop of cultivation of the whole world, is one of vegetables of Chinese cultivated area maximum.Cucumber black spot [Cucumber alternaria leaf spot; Alternaria cucumerina] be grew up in recent years a kind of by the melon alternaric bacteria serious fungal disease that causes, causes harm, become one of important disease of cucumber protection ground, outdoor cropping at present.Be commonly called as roasting leaf disease, burn leaf disease, seedling, strain all can be fallen ill.Main harm blade, general first from cucumber, lower blade starts to occur, then gradually to vertical spread, last remaining several, top greenery only, diseased plant is like the roasting shape of fire.Scab majority is along mesophyll tissue's development of vein both sides, and master pulse is not generally injured.A plurality of scabs when serious of falling ill join together, and mesophyll tissue is withered, and leaf margin scrolls up, and leaf is shrivelled, but does not come off.Cucumber black spot generally in mid-April to May, occur the middle ten days, sickness rate is up to more than 60%, the underproduction 10～20%.The morbidity of knot melon phase, if control not in time, can cause total crop failure, causes tremendous economic loss to plantation family.Especially in today of vegetables high industrial, the breeding for disease resistance pattern based on traditional is seriously restricting the update of cucumber variety, the paces of production industrialization, utilizes modern biotechnology means to carry out the disease-resistant molecular mark of cucumber imperative.
The vegetable thremmatology that is applied as of molecule marker has been opened up brand-new research and Application Areas, and it makes vegetable breeding scholar directly from molecular level, understand the difference between species, also makes breeding process more directly perceived, and purpose is stronger.Molecular marker assisted selection (Molecular marker-assisted selection, MAS) technology has become focus and the important means of breeding research at present, molecular genetic techniques is applied in the research of vegetable disease-resistant genetic breeding, for the selection of material provides brand-new means.Can to showing good genotype, identify in generation morning, can select objective trait seedling stage on DNA level, Disease Resistance Identification can complete indoor, shorten to 2 days qualification cycle, and reliable and stable, be not subject to the impact of the factors such as season, envrionment conditions, can improve selection speed, strengthen accuracy and the reliability selected, accelerate breeding process, therefore have broad application prospects.
Along with the raising of the intensive production of vegetables and cultivation technique, cucumber breeding for disease resistance has been mentioned critical positions.Cucumber black spot is the important disease on cucumber production.Consult domestic and foreign literature, up to now, about the molecular mark research of cucumber black spot still lacks systematic research and report.Thereby the anti-black spot gene of cucumber is combined with the fine quality gene of other kind of anti-black spot not, cultivating good disease-resistant new breeds of cucumbers and creating cucumber new germ plasm is the practical problems of needing solution in production practice badly.Screening and the closely linked molecule marker of the anti-black spot gene of cucumber, obtain, sense black spot gene closely linked gene fragment anti-with cucumber, set up the anti-black spot molecular mark of cucumber system, will promote traditional " experience breeding " to " precise breeding " transformation efficiently, promote breeding efficiency and state of the art, accelerate the process of breeding for disease resistance, for cucumber is hereditary and breeding work lays the foundation.
Summary of the invention
The object of the present invention is to provide the primer sequence for the identification of resistance of cucumber against alternaria cucumerina.
Second object of the present invention is to provide a kind of and closely linked gene fragment of the anti-black spot gene of cucumber.
The 3rd object of the present invention is to provide a kind of and closely linked gene fragment of cucumber sense black spot gene.
Last object of the present invention is to provide a kind of method of identifying resistance of cucumber against alternaria cucumerina.
Technical scheme of the present invention is summarized as follows:
For the identification of the primer sequence of resistance of cucumber against alternaria cucumerina, it is comprised of upstream primer and downstream primer, and upstream primer is the nucleotide sequence described in SEQ ID No1 in sequence table, and downstream primer is the nucleotide sequence described in SEQ ID No2 in sequence table.
With the closely linked gene fragment of the anti-black spot gene of cucumber, it is the nucleotide sequence described in SEQ ID No.3.
With the closely linked gene fragment of cucumber sense black spot gene, it is the nucleotide sequence described in SEQ ID No.4.
A method of identifying resistance of cucumber against alternaria cucumerina, comprises the steps:
(1) extract identified sample Cucumber germplasm DNA;
(2) take described Cucumber germplasm DNA is template, with nucleotides sequence described in SEQ ID No.1 and SEQ ID No.2, classifies upstream and downstream primer as, carries out pcr amplification;
(3) pcr amplification product step (2) being obtained carries out gel electrophoresis, and gel is observed, taken a picture after cma staining under visible ray;
(4), according to the relative position of each identified sample band on gel, identify the resistance of cucumber black spot: the material of the nucleotide sequence described in SEQ ID No.3 appears in all amplified productions, is anti-black spot resource; There is the material of the nucleotide sequence described in SEQ ID No.4 and there is nucleotide sequence described in SEQ ID No.3 and the material of the nucleotide sequence described in SEQ ID No.4 simultaneously, be sense black spot resource in all amplified productions; To the anti-black spot germ plasm resource of cucumber or cucumber is carried out to anti-black spot molecular mark.
The anti-black spot gene fragment of cucumber of utilizing the primer sequence of evaluation resistance of cucumber against alternaria cucumerina of the present invention and amplifying and cucumber sense black spot gene fragment are carried out black spot evaluation of resistance to the F2 building for segregating population and Cucumber Germplasm, its result and artificial inoculation identify that coincidence rate reaches respectively 95.60%, 93.42%, carry out resource resistance screening and have very large using value in breed cucumber.Take these sequences as foundation, can carry out anti-black spot marker assisted selection, breeding, as the probe that detects the anti-black spot gene of cucumber in breed cucumber; Can be for carrying out the anti-black spot assignment of genes gene mapping, mapping and clone, the disease resistance that improves cucumber variety by genetically engineered lays the foundation.
Accompanying drawing explanation
Fig. 1 be utilize to identify that the method for resistance of cucumber against alternaria cucumerina obtains with the closely linked gene fragment of the anti-black spot gene of cucumber and the closely linked gene fragment electrophorogram of sense black spot gene:
A is with the closely linked gene fragment of the anti-black spot gene of cucumber (shown in SEQ ID No.3,122bp);
B is for (SEQ ID No.4 shows, 126bp) with the closely linked gene fragment of cucumber sense black spot gene;
1,4,7,8,10,11 is disease-resistant individual plant; 2,3,5,6,9 is susceptible individual plant, and the resistant strain that isozygotys only has resistant gene fragment, and the perceptual strain of isozygotying only has perceptual gene fragment.
Fig. 2 utilizes a kind of method of identifying resistance of cucumber against alternaria cucumerina, and the maternal L63 of the cucumber sense black spot of acquisition and anti-black spot male parent L9 filial generation F2 are for separation case collection of illustrative plates: 1,2,7,9,10,12,16,18,21,24,26,37,44,48,50,55,65,67,68,70,75,82,83,85 is disease-resistant individual plant; 3,6,13,17,20,28,31,42,43,46,49,52,58,59,60,62,63,64,69,71,76,80 is susceptible individual plant; 4,5,8,11,14,15,19,22,23,25,27,29,30,32～36,38～41,45,47,51,53,54,56,57,61,66,72～74,77～79,81,84,86 is heterozygosis type individual plant, M is DNA standard, the resistant strain that isozygotys only has resistant gene fragment, the perceptual strain of isozygotying only has perceptual gene fragment, and heterozygosis type individual plant has resistant gene fragment and perceptual gene fragment simultaneously.
Fig. 3 utilizes a kind of method of identifying resistance of cucumber against alternaria cucumerina, Cucumber Germplasm is carried out to black spot resistance detection: A for the closely linked gene fragment of the anti-black spot gene of cucumber (shown in SEQ ID No.3,122bp), B is with the closely linked gene fragment of cucumber sense black spot gene (shown in SEQ ID No.4,126bp); 51,52,89～92,102,104,127～129,131,135～137,144,151 is disease-resistant germ plasm resource; 1～50,53～70,72～88,93～101,103,105～126,130,132～134,138～141,143,145～149,152～189 is susceptible germ plasm resource; 71,142,150 is heterozygosis type germ plasm resource.The disease-resistant germ plasm resource of isozygotying only has resistant gene fragment, and the susceptible germ plasm resource of isozygotying only has perceptual gene fragment, and heterozygosis type germ plasm resource has resistant gene fragment and perceptual gene fragment simultaneously.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, and the following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
(1) extract identified sample Cucumber germplasm DNA;
(2) take described Cucumber germplasm DNA is template, with nucleotides sequence described in SEQ ID No.1 and SEQ ID No.2, classify upstream and downstream primer as, carry out pcr amplification, in the special-purpose thin-walled tube of pcr amplification, put into: the nucleotide sequence 25ng described in described Cucumber germplasm DNA20ng, sequence table SEQ ID No1, nucleotide sequence 25ng, dNTP0.20mM, the Mg described in sequence table SEQ ID No2
2+1.5mM, PCR damping fluid, Taq archaeal dna polymerase 1.0 units of 1 times, add aseptic double distilled water to 20 μ l, the special-purpose thin-walled tube of described pcr amplification is put into PCR instrument to increase, amplification condition is: 94 ℃ of denaturations 180 seconds, 94 ℃ of sex change 60 seconds, 51 ℃ of annealing 60 seconds, 72 ℃ are extended 120 seconds, 35 circulations, then 72 ℃ extended 350 seconds, amplification completes;
(3) gel electrophoresis analysis of pcr amplification product: the pcr amplification product that step (2) is obtained adopts 5% denaturing polyacrylamide gel to carry out electrophoretic separation; In amplified production, add denaturant gel sample-loading buffer, be splined on the denaturing polyacrylamide gel of 30 minutes prerunnings, the permanent power electrophoresis of 70W has just arrived the other end of gel to bromjophenol blue, gel is observed, taken a picture after cma staining under visible ray;
(4), according to the relative position of each identified sample band on gel, identify the resistance of cucumber black spot.Because disease-resistant individual plant, susceptible individual plant or intermediate type individual plant produce the DNA fragmentation of different molecular weight after increasing, when separated on gel, its rate of migration on gel is different, thereby the band there are differences on formation position, relative position by band on gel just can carry out the material that the nucleotide sequence described in SEQ ID No.3 appears in all amplified productions of Rapid identification to the resistance of cucumber black spot material, is anti-black spot resource; There is the material of the nucleotide sequence described in SEQ ID No.4 and there is nucleotide sequence described in SEQ ID No.3 and the material of the nucleotide sequence described in SEQ ID No.4 simultaneously, be sense black spot resource in all amplified productions; To the anti-black spot germ plasm resource of cucumber or cucumber is carried out to anti-black spot molecular mark.
Identify Cucumber Germplasm black spot resistance
According to the method for embodiment 1, different Cucumber Germplasm genomic dnas are carried out to pcr amplification, there is the nucleotide sequence described in 122bp(SEQ ID No.3 in all amplified productions) the material of specific fragment, be anti-black spot resource; There is the nucleotide sequence described in 126bp(SEQ ID No.4 in all amplified productions) the material of specific fragment, be sense black spot resource; All materials that simultaneously amplifies these two specific fragments, for impure condensation material, are shown in Fig. 3.Anti-black spot resource, or the carrier of anti-black spot gene, can be as material and the breeding germplasm of further disease-resistant variety seed selection.
Claims (4)
1. for the identification of the primer sequence of resistance of cucumber against alternaria cucumerina, it is characterized in that it is comprised of upstream primer and downstream primer, upstream primer is the nucleotide sequence described in SEQ ID No1 in sequence table, and downstream primer is the nucleotide sequence described in SEQ ID No2 in sequence table.
2. with the closely linked gene fragment of the anti-black spot gene of cucumber, it is characterized in that the nucleotide sequence described in SEQ ID No.3.
3. with the closely linked gene fragment of cucumber sense black spot gene, it is characterized in that the nucleotide sequence described in SEQ ID No.4.
4. identify a method for resistance of cucumber against alternaria cucumerina, it is characterized in that comprising the steps:
(1) extract identified sample Cucumber germplasm DNA;
(2) take described Cucumber germplasm DNA is template, with nucleotides sequence described in SEQ ID No.1 and SEQ ID No.2, classifies upstream and downstream primer as, carries out pcr amplification;
(3) pcr amplification product step (2) being obtained carries out gel electrophoresis, and gel is observed, taken a picture after cma staining under visible ray;
(4), according to the relative position of each identified sample band on gel, identify the resistance of cucumber black spot: the material of the nucleotide sequence described in SEQ ID No.3 appears in all amplified productions, is anti-black spot resource; There is the material of the nucleotide sequence described in SEQ ID No.4 and there is nucleotide sequence described in SEQ ID No.3 and the material of the nucleotide sequence described in SEQ ID No.4 simultaneously, be sense black spot resource in all amplified productions; To the anti-black spot germ plasm resource of cucumber or cucumber is carried out to anti-black spot molecular mark.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105603119A (en) * | 2016-03-31 | 2016-05-25 | 天津科润农业科技股份有限公司 | SNP (single-nucleotide polymorphism) marker method for detecting cucumber Corynespora-cassiicola-resistant site |
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| CN101845504A (en) * | 2010-06-11 | 2010-09-29 | 天津科润农业科技股份有限公司 | Primer sequence for identifying resistance of cucumber against alternaria cucumerina and identification method thereof |
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| CN101845504A (en) * | 2010-06-11 | 2010-09-29 | 天津科润农业科技股份有限公司 | Primer sequence for identifying resistance of cucumber against alternaria cucumerina and identification method thereof |
Non-Patent Citations (2)
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| 孙玉河，等: "《SCAR标记与人工接种鉴定黄瓜种质黑斑病抗性》", 《中国园艺文摘》, 31 July 2012 (2012-07-31) * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603119A (en) * | 2016-03-31 | 2016-05-25 | 天津科润农业科技股份有限公司 | SNP (single-nucleotide polymorphism) marker method for detecting cucumber Corynespora-cassiicola-resistant site |
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