CN103555857B - 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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- CN103555857B CN103555857B CN201310576852.0A CN201310576852A CN103555857B CN 103555857 B CN103555857 B CN 103555857B CN 201310576852 A CN201310576852 A CN 201310576852A CN 103555857 B CN103555857 B CN 103555857B
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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 the technique and method of cucumber conventional cross-breeding, molecular mark.Particularly with cucumber the closely linked gene fragment of anti-black spot gene, with the closely linked gene fragment of cucumber sense black spot gene, qualification resistance of cucumber against alternaria cucumerina method.
Background technology
Cucumber (Cucumis Sativus L.) is Curcurbitaceae (Cucurbitaceae) Cucumis (Cucumis) 1 year herbaceous plant that overgrows, and is the vegetables crop generally cultivated of the whole world, is one of maximum vegetables of Chinese cultivated area.Cucumber black spot [Cucumber alternaria leaf spot; Alternaria cucumerina] be that grew up in recent years a kind of is caused by melon alternaric bacteria, serious fungal disease of causing harm, become one of the important disease of cucumber protecting field, outdoor cropping at present.Be commonly called as roasting leaf disease, burn leaf disease, seedling, strain all can fall ill.Main harm blade, general first from generation in cucumber, lower blade, then gradually to vertical spread, be finally only left several, top greenery, diseased plant bakes shape like fire.The most mesophyll tissue along vein both sides of scab develops, and master pulse is not generally injured.When falling ill serious, multiple scab joins together, and mesophyll tissue is withered, and leaf margin scrolls up, and leaf is shrivelled, but does not come off.Cucumber black spot generally mid-April on May, the middle ten days occur, sickness rate 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 to lose to plantation family.Especially in today of vegetables high industrial, seriously govern the update of cucumber variety, the paces of production industrialization based on traditional breeding for disease resistance pattern, utilize modern biotechnology means to carry out the disease-resistant molecular mark of cucumber imperative.
The vegetable thremmatology that is applied as of molecule marker opens brand-new investigation and application field, and it makes vegetable breeding scholar directly can understand the difference between species from molecular level, and also make breeding process more directly perceived, 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 be applied in the research of vegetable disease-resistant genetic breeding, the selection for material provides brand-new means.Can early identify for the excellent genotype of performance, seedling stage can be selected objective trait on DNA level, Disease Resistance Identification can complete in indoor, shorten to 2 days qualification cycle, and reliable and stable, not by the impact of the factor such as season, envrionment conditions, selection speed can be improved, strengthen the accuracy and reliability selected, accelerate breeding process, therefore have broad application prospects.
Along with the raising of vegetables intensive manufacture and cultivation technique, cucumber breeding for disease resistance mentions 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.Thus combined by the fine quality gene of anti-for cucumber black spot gene with other kind of not anti-black spot, new breeds of cucumbers and the creation cucumber new germ plasm of cultivating excellent resistant are the practical problemss needing solution in production practice badly.Screening and the closely linked molecule marker of cucumber anti-black spot gene, obtain and resist with cucumber, feel the closely linked gene fragment of black spot gene, set up cucumber anti-black spot molecular mark system, traditional " experience breeding " will be promoted to " precise breeding " transformation efficiently, promote breeding efficiency and state of the art, accelerate the process of breeding for disease resistance, for cucumber heredity and breeding work lay 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 the closely linked gene fragment of cucumber anti-black spot gene.
3rd object of the present invention is to provide a kind of and the closely linked gene fragment of cucumber sense black spot gene.
Last object of the present invention is to provide a kind of method 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 made up of upstream primer and downstream primer, and upstream primer is the nucleotide sequence in sequence table described in SEQ ID No1, and downstream primer is the nucleotide sequence in sequence table described in SEQ ID No2.
With the closely linked gene fragment of cucumber anti-black spot gene, 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.
Identify a method for resistance of cucumber against alternaria cucumerina, comprise the steps:
(1) identified sample Cucumber germplasm DNA is extracted;
(2) with described Cucumber germplasm DNA for template, arrange as upstream and downstream primer with nucleotides sequence described in SEQ ID No.1 and SEQ ID No.2, carry out pcr amplification;
(3) pcr amplification product that step (2) obtains carried out gel electrophoresis, gel is observed under visible light after cma staining, take a picture;
(4) according to the relative position of each identified sample band on gel, the resistance of qualification 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 the material of the nucleotide sequence simultaneously had described in SEQ ID No.3 and the nucleotide sequence described in SEQ ID No.4 in all amplified productions, is sense black spot resource; Anti-black spot molecular mark is carried out to cucumber anti-black spot germ plasm resource or to cucumber.
The primer sequence utilizing qualification resistance of cucumber against alternaria cucumerina of the present invention and the anti-black spot gene fragment of the cucumber amplified thereof and cucumber sense black spot gene fragment carry out black spot evaluation of resistance to the F2 built for segregating population and Cucumber Germplasm, its result and artificial infection idenfication coincidence rate reach 95.60%, 93.42% respectively, carry out resource resistance screening and have very large using value in breed cucumber.With these sequences for foundation, anti-black spot marker assisted selection, breeding can be carried out, as the probe detecting the anti-black spot gene of cucumber in breed cucumber; For carrying out the anti-black spot assignment of genes gene mapping, mapping and clone, can be laid the foundation by the disease resistance of genetically engineered improvement cucumber variety.
Accompanying drawing explanation
Fig. 1 be utilize the method for qualification resistance of cucumber against alternaria cucumerina to obtain with the closely linked gene fragment of cucumber anti-black spot gene and feel the closely linked gene fragment electrophorogram of black spot gene:
A for the closely linked gene fragment of cucumber anti-black spot gene (shown in SEQ ID No.3,122bp);
B is and the closely linked gene fragment of cucumber sense black spot gene (SEQ ID No.4 shows, 126bp);
1,4,7,8,10,11 is disease-resistant individual plant; 2,3,5,6,9 is susceptible individual plant, and homozygous resistant strain only has resistance gene fragment, and perceptual strain of isozygotying only has perceptual gene fragment.
Fig. 2 utilizes a kind of method identifying resistance of cucumber against alternaria cucumerina, and the maternal L63 and anti-black spot male parent L9 filial generation F2 of cucumber sense black spot of acquisition is 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 hybrid type individual plant, M is DNA standard, homozygous resistant strain only has resistance gene fragment, perceptual strain of isozygotying only has perceptual gene fragment, and hybrid type individual plant has resistance gene fragment and predisposing genes fragment simultaneously.
Fig. 3 utilizes a kind of method identifying resistance of cucumber against alternaria cucumerina, black spot Resistance detecting is carried out to Cucumber Germplasm: A is for the closely linked gene fragment of cucumber anti-black spot gene (shown in SEQ ID No.3,122bp), B for 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 Resistant gerplasm 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 hybrid type germ plasm resource.Resistant gerplasm resource of isozygotying only has resistance gene fragment, and susceptible germ plasm resource of isozygotying only has perceptual gene fragment, and hybrid type germ plasm resource has resistance gene fragment and predisposing genes 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.
Embodiment 1
(1) identified sample Cucumber germplasm DNA is extracted;
(2) with described Cucumber germplasm DNA for template, arrange as upstream and downstream primer with nucleotides sequence described in SEQ ID No.1 and SEQ ID No.2, carry out pcr amplification, put in the special thin-walled tube of pcr amplification: described Cucumber germplasm DNA20ng, the nucleotide sequence 25ng described in 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 unit of 1 times, add aseptic double distilled water to 20 μ l, special for described pcr amplification thin-walled tube is put into PCR instrument increase, amplification condition is: 94 DEG C of denaturations 180 seconds, 94 DEG C of sex change 60 seconds, 51 DEG C of annealing 60 seconds, 72 DEG C extend 120 seconds, 35 circulations, then 72 DEG C extend 350 seconds, amplification completes;
(3) gel electrophoresis analysis of pcr amplification product: adopt 5% denaturing polyacrylamide gel to carry out electrophoretic separation the pcr amplification product that step (2) obtains; Denaturant gel sample-loading buffer is added in amplified production, be splined on the denaturing polyacrylamide gel of 30 minutes prerunnings, 70W invariable power electrophoresis has just arrived the other end of gel to bromjophenol blue, gel is observed under visible light after cma staining, take a picture;
(4) according to the relative position of each identified sample band on gel, the resistance of qualification 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 gel is separated, its rate of migration on gel is different, thus the band that forming position there are differences, just can carry out to the resistance of cucumber black spot material the material that the nucleotide sequence described in SEQ ID No.3 appears in all amplified productions of Rapid identification by the relative position of band on gel, be anti-black spot resource; There is the material of the nucleotide sequence described in SEQ ID No.4 and the material of the nucleotide sequence simultaneously had described in SEQ ID No.3 and the nucleotide sequence described in SEQ ID No.4 in all amplified productions, is sense black spot resource; Anti-black spot molecular mark is carried out to cucumber anti-black spot germ plasm resource or to cucumber.
Embodiment 2
Qualification Cucumber Germplasm black spot resistance
According to the method for embodiment 1, carrying out pcr amplification to different Cucumber Germplasm genomic dna, 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 simultaneously amplifying these two specific fragments, are impure condensation material, see Fig. 3.Anti-black spot resource, or the carrier of anti-black spot gene, can be used as material and the breeding kind matter of further disease-resistant variety seed selection.
Claims (2)
1., with the closely linked gene fragment of cucumber anti-black spot gene, it is characterized in that the nucleotide sequence described in SEQIDNo.3.
2., with the closely linked gene fragment of cucumber sense black spot gene, it is characterized in that the nucleotide sequence described in SEQIDNo.4.
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