CN104789703A - Method for rapid evaluation of variety resistance to bushy stunt of wheat by detecting proliferation rate of virus in coleoptiles - Google Patents
Method for rapid evaluation of variety resistance to bushy stunt of wheat by detecting proliferation rate of virus in coleoptiles Download PDFInfo
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Abstract
The invention discloses a method for rapid evaluation of variety resistance to bushy stunt of wheat by detecting proliferation rate of virus in coleoptiles. The method comprises the following steps: 1) selecting identification scale; 2) performing hypoxia induction on wheat to be detected and identifying the coleoptiles of scale variety; 3) inoculating viruliferous laodelphax striatellus into the coleoptiles and then performing dark culture; 4) performing fluorescence quantitative PCR (polymerase chain reaction) to obtain relative expression (RQ value) of RBSDV (rice black streaked dwarf virus) virus S7 and an internal control gene of various wheat varieties at period time points; 5) drawing an RBSDV proliferation index trend line; and 6) evaluating the resistance level and the resistance grade of the wheat variety to be detected to the bushy stunt of the wheat according to the RBSDV proliferation index trend line formula. According to the method disclosed by the invention, the variety resistance to the bushy stunt of the wheat is rapidly evaluated by detecting the proliferation rate of the virus in the coleoptiles, the resistance level and the resistance grade of the wheat variety to the bushy stunt of the wheat can be rapidly determined on molecular scale, the identification period is short and the reliability is high, thereby having relatively great breeding application value.
Description
Technical field
The present invention relates to a kind of method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance that qualification cycle is short, reliability is high, belonging to Plant diseases Resistance Identification technology.
Background technology
Wheat is the general designation of Triticum plant, is a kind of grass in extensively plantation all over the world, originates from the crescent fertile soil area in the Middle East the earliest.Wheat is one of three large cereal, and the overwhelming majority, as edible, only about has 1/6th to use as feed.Wheat is also Chinese second largest food crop as the food crop of ultimate production second in the world, in compatriots' food, play key player.According to statistics, in the grain ration total quantity consumed of China, wheat accounts for about 43%.In recent years, world population increases and people's living standard improves constantly, grain demand sustainable growth, meanwhile, plough reduce gradually, water resources is not enough, wheat diseases takes place frequently, the deterioration of the ecological environment, natural disaster take place frequently, and feed-use grain and the increase of biofuel consumption grain etc. bring stern challenge to world food supply, Wheat Production becomes more and more important in the status of grain security.
Wheat rosette stunt flies louse-borne virus disease by passing malicious Medium Ash, and be that the one of northwest and North China often sends out venereal disease evil, the classical symptom of wheat rosette stunt shows as blade yellowish green alternate striped, tillers and increases, and plant is downgraded, and forms the short shape of obvious clump.Before winter, susceptible strain major part can not be survived the winter and dead, and before the winter, after aobvious disease and early spring jointing, disease plant upper blade shows striped, generally can not ear and withered ahead of time, some can heading, but fringe fine grain is not plump thin, larger to yield effect.Wheat rosette stunt is distributed more widely in China, and (district, city) is economized in Shaanxi, Gansu, Ningxia, the Inner Mongol, Shandong, Shanxi, Hebei, Henan, Jiangsu, Zhejiang, Xinjiang, Beijing, Tianjin etc. all generation, and the degree that causes harm is different because of time and area.The hypopathia field underproduction 10% ~ 20%, the grave illness field underproduction more than 50%, even has no harvest.
Wheat rosette stunt virus passes poison primarily of small brown rice planthopper.At the most northern area of China, there are for 3 ~ 4 generations in small brown rice planthopper 1 year, 2 ~ 3 age nymph survive the winter in weeds rhizosphere or soil seam.Mid-March in next year comes into play, the weeds tender leaf that concentrated harm is newly turned green.Early April, go to wheatland by ridge after winter wheat turns green or spring wheat emerges, the population density by ridge is high, after gradually spread to Tanaka.Late April is the winter generation adult emergence Sheng phase.Late June first-generation adult appearance.After wheat maturation, small brown rice planthopper goes to autumn crop and limit, field weeds, then breeds for 1 ~ 2 generation, moves under the weeds clumps such as ridge survive the winter in soil November.Small brown rice planthopper 1 ~ 2 nymph in age easily obtains poison, and it is the strongest that adult passes malicious ability, the shortlyest obtains poison time 12h, and most short pass poison time 20min, obtains malicious rate and pass malicious rate and improve with sucking time lengthening.Small brown rice planthopper can be with poison throughout one's life once obtain poison, but not transovarian transmission.Virus is survived the winter in nymph body.In Winter Wheat Area, small brown rice planthopper migrates in a large number to wheatland autumn from viruliferous more summer host, causes the seedling morbidity of early sowing autumn.The malicious nymph of band that survives the winter is malicious source, moves back to wheatland harm next year.In recent years, along with wheat paddock small brown rice planthopper generation quantity rises year by year, the occurrence and harm degree of wheat rosette stunt is also more and more serious, has become the serious plant disease of restriction China Wheat Production, has brought great challenge to the grain security of China.
At present, mainly use pesticide control to pass virus mediator small brown rice planthopper to the control of wheat rosette stunt, but cause greatly prevention effect not good due to amboceptor the quantity of insects' population, and there is environmental pollution.Cultivating disease-resistant variety is the most economic, the effective means of all kinds of disease of control.Screening, excavation and innovation Resistant gerplasm are prerequisite and the basis of carrying out breeding for disease resistance, and resistant gene location and clone, then for utilizing marker assisted selection equimolecular breeding technique, quickening and high effect culture disease-resistant variety to provide brand-new and favourable instrument.But wheat rosette stunt Resistance QTL detection so far and genetic effect analysis are also in the starting stage, the kind to wheat rosette stunt immunity is not found, so carry out Large-scale Screening to wheat rosette stunt resistance kind matter and launch wheat rosette stunt genetics of resistance characteristic research seeming particularly urgent on producing yet.
It is the most direct effective means that screening Resistant germplasm is applied to breeding for disease resistance production, current wheat breeding screens disease-resistant variety, localization of disease resistance genes qualification resistance family many employings field natural occurrence identification method and artificial inoculation pass virus mediator identification method, but the qualification of natural appraisal method can not get rid of the interference of driving the sick qualification result of sex resistance, and artificial inoculation passes the demand that virus mediator identification method is difficult to the scale amount meeting varietal resistance qualification required biography virus mediator, and field natural occurrence qualification and artificial inoculation pass virus mediator qualification all to be needed by just carrying out phenotypic evaluation after wheat Overwintering Growth, qualification cycle is very long, limit applying of the method.
Scientific technological advance makes rapid progress, technology for test sensitivity plant virus also develops rapidly, except traditional biological method, serological method, electron microscope method, chip detection method, the new molecular biology method risen provides new approaches to the qualification of plant virus.Molecular biology method mainly detects kind and the existence of virus by the nucleic acid (DNA, RNA) extracting virus.The qualitative detection molecular biology method of main application comprises viral nucleic acid molecule hybridization technique, dsRNA electrophoretic technique and polymerase chain reaction (PCR) technology at present, and detection by quantitative mainly refers to Real-Time Fluorescent Quantitative PCR Technique.This kind of method is highly sensitive, high specificity, and detection speed is fast, operating process also easier, can be used for a large amount of sample detection.Quantitative fluorescent PCR, can detection by quantitative viral copy number in plant virus Detection and Identification.During application fluorescence quantitative PCR detection unknown sample, compare that other quantivative approachs and regular-PCR method specificity are stronger, accuracy is high, highly sensitive, linear relationship is wide, safety simple to operate, the pollution of sample comparatively the end (amplification and the detection of sample are carried out in same pipe), and does not need post-processed.Because fluorescence quantitative PCR method is to the superiority detecting unknown sample content, at numerous areas as plant pathology Mechanism Study, the target gene expression level in transgenic research and clinical medicine context of detection obtain extensive and ripe application.
We study and find wheat plant inoculation RBSDV, no matter be in disease-resistant or susceptible wheat plant body, RBSDV virus all detected, after wheat plant infects RBSDV virus seedling stage in addition, symptom does not highlight, do not determine its disease resistance by phenotype, after needing to wait until aobvious disease of surviving the winter, its disease resistance could be determined.Stem-winding, we study recently, and to find that wheat hypoxemia sprouts the RBSDV virus multiplication speed that the coleoptile produced inoculates after RBSDV virus in for some time coleoptile directly related with wheat breed resistance.Coleoptile is the taper shell-like thing outside wheatgerm, and be a sheath-like structure, coleoptile was the protective tissue of wheat leaf blade originally, has the effect of more immature leaf and growing tip in protection plumule.Usual wheat plant infects RBSDV virus and all passes poison at Wheat Seedling by small brown rice planthopper, therefore the biography poison research that seed germination produces coleoptile is left in the basket, we study and find that the RBSDV virus multiplication speed after coleoptile inoculation RBSDV virus in for some time coleoptile is directly related with wheat breed resistance, and this provides new approaches to the Resistance Identification of wheat rosette stunt.
The present invention cultivates by the suppression of hypoxia inducible and coleoptile late stage of culture hormone that coleoptile cultivates hormone in early stage the growth time expanding coleoptile, and at this moment in detected by the RBSDV content in coleoptile after fluorescence quantitative RT-RCR interface differential technique kind virus, achieve by making RBSDV propagation trend map the RBSDV virus multiplication speed in the rear coleoptile of poison that connects to measure quickly and accurately, and then Rapid identification wheat breed is to the resistance level of wheat rosette stunt and resistance class.
Present method is utilized to evaluate wheat breed to the resistance level of wheat rosette stunt, both the interference of antixenosis to qualification result can have been removed, overcome again the defect that the general survey method cycle is oversize, accuracy is low, by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance, there is the advantage that qualification cycle is short, reliability is high.This authentication method, except can being applied to the excavation qualification of wheat variety resources, varietal resistance evaluation, disease-resistant variety seed selection, can also be applied to the various fields such as phenotypic evaluation and genetics of resistance law study of localization of disease resistance genes.
Summary of the invention
The object of the invention is, for current wheat rosette stunt varietal resistance authentication method, there is long, the shortcoming such as qualification result is inaccurate qualification cycle, providing a kind of method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance that qualification cycle is short, reliability is high.
The object of the invention is to solve by the following technical programs:
By detecting a method for coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance, it is characterized in that: this authentication method comprises the following steps:
1) qualification scale is selected: according to manually connecing different varieties in poison qualification, high resistance, middle resistant, susceptible three qualification scales being arranged to wheat rosette stunt performance, selecting three corresponding wheat breeds as qualification scale;
2) coleoptile of hypoxia inducible wheat to be measured and qualification scale kind: each kind gets 15-20 grain seed, soak 15min with 0.6% chlorine bleach liquor and carry out sterilizing, ultrapure water is clean, then be positioned over hypoxia inducible in the IAA solution of suitable concentration under 25 DEG C of conditions and cultivate 3 days, induce coleoptile;
3) light culture after the malicious small brown rice planthopper of coleoptile inoculation band: coleoptile ultrapure water is cleaned, put into the test tube being covered with absorbent wool, each test tube seed, the malicious small brown rice planthopper of quantitative inoculation band, by horizontal after breathable gauze closed test tube, pass malicious 24h, coleoptile ultrapure water after poison will be passed, light culture on the absorbent wool having infiltrated the ABA solution of suitable concentration under being then positioned over 25 DEG C of conditions;
4) quantitative fluorescent PCR obtains the relative expression quantity (RQ value) of RBSDV virus S7 that each wheat breed selects cycle time and reference gene: according to selecting the coleoptile 40-60g gathering wheat to be measured He identify scale specific position cycle time, the each time point of drawing materials of each kind draw materials 2-3 repeat,-70 DEG C of preservations, extract RNA and purifying, get the above-mentioned viral RNA extracting solution of 1 μ L, add quantitative fluorescent PCR reaction system, utilize primer RBSDV-F:5 '-AGA GCT CTT CTA GTT ATT GCG-3 ' and RBSDV-R:5 '-TCA GCA AAA GGT AAA GGA ACG-3 ' according to fluorescent quantitative PCR program amplification RBSDV virus S7 fragment, increase complete, enter interpretation of result interface, take GAPDH as internal reference, compared with control group, obtain the RBSDV virus S7 of each sampling time point repetition of each kind and the relative expression quantity (RQ value) of reference gene,
5) RBSDV proliferation index Trendline is drawn: utilize each kind to be measured of multiple multiple RQ Data-Statistics and qualification scale to raise the RQ value average of the rear each sampling time point of poison, be ordinate zou with RQ value average, raise the rear cycle sampling time point of poison for X-coordinate, statistical study is carried out to data, obtains RBSDV proliferation index Trendline y=Ae
kx;
6) wheat breed to be measured is evaluated to the resistance level of wheat rosette stunt and resistance class according to RBSDV proliferation index Trendline formula: according to each kind RBSDV proliferation index Trendline formula y=Ae
kxevaluate the resistance level of wheat breed to be measured, namely the kind that the kind that in formula, k value is large is less than k value is low to RBSDV resistance, what kind k value to be measured was less than or equal to high resistance scale is high resistance kind, kind k value to be measured be greater than high resistance scale and in being less than or equal to anti-scale be in anti-kind, anti-scale during kind k value to be measured is greater than and be less than or equal to susceptible scale for susceptible variety, what kind k value to be measured was greater than susceptible scale is telegraphy kind.
Described step 1) and 6) in high resistance identify that scale is that indoor connect during poison is identified and disease-resistantly show that in local wheat variety resources, rank is 9%-11%; In anti-ly identify that scale rank is 23%-27%; Susceptiblely identify that scale rank is 47%-53%.
Described step 2) in the IAA strength of solution of suitable concentration be 1.5-2.5 μm of ol/L; Described hypoxia inducible method is that under seed is placed in liquid level, 5-10cm place cultivates.
The obtaining step of the band poison small brown rice planthopper in described step 3) is:
3.1) RBSDV poison source obtains: gather the tender diseased plant of wheat children showing as wheat rosette stunt symptom from field, wheat rosette stunt grave illness district ,-70 DEG C of preservations;
3.2) virus mediator successive propagation is passed: land for growing field crops gathers and purifying small brown rice planthopper nymph at advanced age, raises and go down to posterity with wheat seedling;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out wheat rosette stunt diseased plant when raising poison, normal temperature places 3-5h makes diseased plant naturally launch, then closely wrap up with plastics bag with after water suction cotton parcel diseased plant root, guarantee that water can not flow out, then moved in crisper, 1.5-2.5 small brown rice planthopper in the age nymph that indoors artificial is raised is proceeded on the RBSDV poison source wheat in crisper, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred on wheat seedling raise spend the phase of walking around to be band malicious small brown rice planthopper.
The ABA strength of solution of the suitable concentration in described step 3) controls as 40-60 μm of ol/L; The malicious small brown rice planthopper of quantitative inoculation band in described step 3) controls as a 6-10 worm/coleoptile.
Periodic sampling time point in described step 4) is that RBSDV passes in malicious latter 4-10 days every a 1.5-2.0 days equally distributed 3-4 time point; The coleoptile of specific position is the position, middle of coleoptile length.
The method of the extraction RNA in described step 4) is: get appropriate blade and add liquid nitrogen and grind rapidly in mortar, add in TRI-Reagent to 1.5ml centrifuge tube in proportion, sample volume should more than TRI-Reagent volume 10%, 4 DEG C of 12000 × g low-temperature centrifugation 1.5min, then carefully shift supernatant in new centrifuge tube.
The method of the purifying RNA in described step 4) is: a) directly add a volume (95%-100%) alcohol to being dissolved in the supernatant liquor of TRI-Reagent, whirlpool mixes; B) transfer in adsorption column and filter (adsorption column is placed in collection tube), the then centrifugal 1min of 12000 × g, afterwards adsorption column is transferred in new collection tube; C) 400 μ l RNA Wash Buffer are added in centrifugal column, the centrifugal 1min of 12000 × g; D) the DNAase I cocktail buffer prepared by 80 μ l is directly added in centrifugal column, 25-37 DEG C of heating in water bath centrifugal column 15min, the centrifugal 30s of 12000 × g; E) add 400 μ l RNA Prewash in adsorption column, the centrifugal 1min of 12000 × g, collect the liquid filtered, repeat this step; F) add 700 μ l RNA Wash Buffer in adsorption column, the centrifugal 1min of 12000 × g, then from collection tube careful transfer adsorption column in a new RNase-free 1.5ml centrifuge tube; G) at least add 25 μ l DNase/RNase-Free Water in centrifuge tube matrix, maximum velocity centrifugation 1min, eluted RNA can directly utilize or be stored in-70 DEG C of Ultralow Temperature Freezers.
Contain in the every 20 μ L of fluorescent quantitation reaction system in described step 4): the ddH of 2 × qPCR master mix (Promega) of 10 μ L, the cDNA template of 1.0 μ L, 8.2 μ L
210 μMs of primer RBSDV-R and RP of O and 0.8 μ L; Real-time fluorescence quantitative PCR amplification program is: 95 DEG C of denaturation 2min; 95 DEG C of sex change 15s, 60 DEG C of annealing 15s, 72 DEG C extend 20s, carry out 40 circulations altogether; 95 DEG C of sex change 15s, 60 DEG C of annealing 1min, 95 DEG C of sex change 15s.
Described step 3.3) in RBSDV poison source wheat derive from step 3.1) in the tender diseased plant of wheat children showing as wheat rosette stunt symptom.
Accompanying drawing explanation
Accompanying drawing is obtained RBSDV proliferation index trend map by draw materials detection RBSDV change in concentration of a little drawing materials of cycle after passing poison to the qualification scale coleoptile selected by embodiment.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
A molecular assay method for wheat rosette stunt, is characterized in that: this authentication method comprises the following steps:
1) qualification scale is selected: according to manually connecing different varieties in poison qualification, high resistance, middle resistant, susceptible three qualification scales are arranged to wheat rosette stunt performance, select three corresponding wheat breeds as qualification scale, wherein, high resistance identifies that scale is that indoor connect during poison is identified and disease-resistantly show that in local wheat variety resources, rank is 9%-11%, in anti-ly identify that scale rank is 18%-22%, susceptiblely identify that scale rank is 47%-53%;
2) coleoptile of hypoxia inducible wheat to be measured and qualification scale kind: each kind gets 15-20 grain seed, soak 15min with 0.6% chlorine bleach liquor and carry out sterilizing, ultrapure water is clean, then under being positioned over the IAA liquid level of solution of 1.5-2.5 μm of ol/L under 25 DEG C of conditions, 5-10cm place hypoxia inducible cultivates 3 days, induces coleoptile;
3) light culture after the malicious small brown rice planthopper of coleoptile inoculation band:
3.1) RBSDV poison source obtains: gather the tender diseased plant of wheat children showing as wheat rosette stunt symptom from field, wheat rosette stunt grave illness district ,-70 DEG C of preservations;
3.2) virus mediator successive propagation is passed: land for growing field crops gathers and purifying small brown rice planthopper nymph at advanced age, raises and go down to posterity with wheat seedling;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out wheat rosette stunt diseased plant when raising poison, normal temperature places 3-5h makes diseased plant naturally launch, then closely wrap up with plastics bag with after water suction cotton parcel diseased plant root, guarantee that water can not flow out, then moved in crisper, 1.5-2.5 small brown rice planthopper in the age nymph that indoors artificial is raised is proceeded on the RBSDV poison source wheat in crisper, this RBSDV poison source wheat that is 3.1) in the tillering regularity wheat diseased plant showing as wheat rosette stunt symptom, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred on wheat seedling raise spend the phase of walking around to be band malicious small brown rice planthopper,
3.4) the malicious small brown rice planthopper of coleoptile inoculation band: coleoptile ultrapure water is cleaned, put into the test tube being covered with absorbent wool, each test tube seed, quantitatively inoculate the malicious small brown rice planthopper of band according to the ratio of a 6-10 worm/coleoptile, by horizontal after breathable gauze closed test tube, pass malicious 24h;
3.5) coleoptile connects light culture after poison: by coleoptile ultrapure water, light culture on the absorbent wool having infiltrated the ABA solution of 40-60 μm of ol/L under being then positioned over 25 DEG C of conditions;
4) quantitative fluorescent PCR obtains the relative expression quantity (RQ value) of RBSDV virus S7 that each wheat breed selects cycle time and reference gene: small brown rice planthopper passes maliciously terminates rear 4-10 days inherences and gather wheat to be measured every a 1.5-2.0 days equally distributed 3-4 time point and identify position, the middle 40-60g of scale coleoptile length, the each time point of drawing materials of each kind draw materials 2-3 repeat,-70 DEG C of preservations, extract RNA and purifying, (RNA extraction method is: get appropriate blade and add liquid nitrogen and grind rapidly in mortar, add in TRI-Reagent to 1.5ml centrifuge tube in proportion, sample volume should more than TRI-Reagent volume 10%, 4 DEG C of 12000 × g low-temperature centrifugation 1.5min, then supernatant is carefully shifted in new centrifuge tube, RNA purification process is: a) directly add a volume (95%-100%) alcohol to being dissolved in the supernatant liquor of TRI-Reagent, whirlpool mixes, b) transfer in adsorption column and filter (adsorption column is placed in collection tube), the then centrifugal 1min of 12000 × g, afterwards adsorption column is transferred in new collection tube, c) 400 μ l RNA Wash Buffer are added in centrifugal column, the centrifugal 1min of 12000 × g, d) the DNAase I cocktail buffer prepared by 80 μ l is directly added in centrifugal column, 25-37 DEG C of heating in water bath centrifugal column 15min, the centrifugal 30s of 12000 × g, e) add 400 μ l RNA Prewash in adsorption column, the centrifugal 1min of 12000 × g, collect the liquid filtered, repeat this step, f) add 700 μ l RNA Wash Buffer in adsorption column, the centrifugal 1min of 12000 × g, then from collection tube careful transfer adsorption column in a new RNase-free 1.5ml centrifuge tube, g) at least add 25 μ l DNase/RNase-Free Water in centrifuge tube matrix, maximum velocity centrifugation 1min, eluted RNA can directly utilize or be stored in-70 DEG C of Ultralow Temperature Freezers.) get the above-mentioned viral RNA extracting solution of 1 μ L, add quantitative fluorescent PCR reaction system, utilize primer RBSDV-F:5 '-AGA GCT CTT CTA GTT ATT GCG-3 ' and RBSDV-R:5 '-TCA GCA AAA GGT AAA GGA ACG-3 ' according to fluorescent quantitative PCR program amplification RBSDV virus S7 fragment, increase complete, enter interpretation of result interface, take GAPDH as internal reference, compared with control group, obtain the RBSDV virus S7 of each sampling time point repetition of each kind and the relative expression quantity (RQ value) of reference gene;
5) RBSDV proliferation index Trendline is drawn: utilize each kind to be measured of multiple multiple RQ Data-Statistics and qualification scale to raise the RQ value average of the rear each sampling time point of poison, be ordinate zou with RQ value average, raise the rear cycle sampling time point of poison for X-coordinate, statistical study is carried out to data, obtains RBSDV proliferation index Trendline y=Ae
kx;
6) wheat breed to be measured is evaluated to the resistance level of wheat rosette stunt and resistance class according to RBSDV proliferation index Trendline formula:
6.1) kind resistance level to be measured is evaluated: according to each kind RBSDV proliferation index Trendline formula y=Ae
kxevaluate the resistance level of wheat breed to be measured, the kind that the kind that namely in formula, k value is large is less than k value is low to RBSDV resistance;
6.2) kind resistance class to be measured divides: the resistance class evaluating kind to be measured according to kind to be measured and qualification scale k value size, namely kind k value to be measured is less than or equal to high resistance scale is high resistance kind, kind k value to be measured be greater than high resistance scale and in being less than or equal to anti-scale be in anti-kind, anti-scale during kind k value to be measured is greater than and to be less than or equal to susceptible scale be susceptible variety, it is telegraphy kind that kind k value to be measured is greater than susceptible scale.
Embodiment
2015, the authentication method of this invention is utilized to carry out bushy stunt Resistance Identification to collecting the part wheat variety resources obtained.Spoke Ah No. 1, A Bo, ripe three wheat breeds of greenery are set respectively as high resistance, middle resistant, susceptible qualification scale, wherein, spoke Ah No. 1 connects in poison qualification in 2013-2014 indoor and disease-resistantly shows rank the 5th, A Bo rank 13 in 51 wheat variety resources colonies, the ripe rank 26 of greenery.Coleoptile acquisition time be set to connect poison complete after the 4th, 8,12,16 day four time points.
Real-time fluorescence quantitative PCR obtains the RQ value of each kind periodic sampling time point, as shown in Table 1:
Then draw RBSDV proliferation index Trendline: with RQ value average for ordinate zou, raising the rear sampling time point of poison is X-coordinate, carries out statistical study to data, obtains RBSDV proliferation index Trendline, as shown in drawings.
Bushy stunt resistance level and the resistance class of wheat breed to be measured is evaluated according to the difference of RBSDV proliferation index trend map each kind k value.In this experiment, high resistance scale spoke Ah No. 1 k value is calculated as 0.7102; In anti-scale A Bo k value be calculated as 1.4010; The ripe k value of susceptible scale greenery is calculated as 2.0001, as shown in drawings.Kind k value to be measured compares obtains corresponding resistance class to qualification scale, and we adopt authentication method of the present invention to identify a kind matter bushy stunt resistance to following 11 wheat breeds, and result is as follows:
By the qualification result of table two is fallen ill comparing of result with field multiple years bushy stunt, we find that this qualification result and land for growing field crops result of falling ill is very identical, and the present invention just can detect accurately kind of a matter bushy stunt resistance in the short period of time after biography virus mediator connects malicious detaehed wheat leaves, thus substantially reduce qualification time.
Can be found by embodiment, new Identification Method of the present invention, compared with authentication method in the past, being evaluated wheat rosette stunt varietal resistance by connecing malicious detaehed wheat leaves " Invest, Then Investigate " coleoptile inner virus rate of propagation, being substantially reduced qualification cycle; Evaluate compared with the method for anti-perception with utilizing illness, determine that wheat germplasm is to the resistance level of wheat rosette stunt and resistance class by detecting coleoptile inner virus rate of propagation from molecular level, reliability is higher, and thus the present invention has larger Breeding Application value.
Above embodiment is only and technological thought of the present invention is described, can not limit protection scope of the present invention with this, every technological thought proposed according to the present invention, and any change that technical scheme basis is done, all falls within scope; The technology that the present invention does not relate to all is realized by prior art.
Claims (10)
1., by detecting a method for coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance, it is characterized in that: this authentication method comprises the following steps:
1) qualification scale is selected: according to manually connecing different varieties in poison qualification, high resistance, middle resistant, susceptible three qualification scales being arranged to wheat rosette stunt performance, selecting three corresponding wheat breeds as qualification scale;
2) coleoptile of hypoxia inducible wheat to be measured and qualification scale kind: each kind gets 15-20 grain seed, soak 15min with 0.6% chlorine bleach liquor and carry out sterilizing, ultrapure water is clean, then be positioned over hypoxia inducible in the IAA solution of suitable concentration under 25 DEG C of conditions and cultivate 3 days, induce coleoptile;
3) light culture after the malicious small brown rice planthopper of coleoptile inoculation band: coleoptile ultrapure water is cleaned, put into the test tube being covered with absorbent wool, each test tube seed, the malicious small brown rice planthopper of quantitative inoculation band, by horizontal after breathable gauze closed test tube, pass malicious 24h, coleoptile ultrapure water after poison will be passed, light culture on the absorbent wool having infiltrated the ABA solution of suitable concentration under being then positioned over 25 DEG C of conditions;
4) quantitative fluorescent PCR obtains the relative expression quantity (RQ value) of RBSDV virus S7 that each wheat breed selects cycle time and reference gene: according to selecting the coleoptile 40-60g gathering wheat to be measured He identify scale specific position cycle time, the each time point of drawing materials of each kind draw materials 2-3 repeat,-70 DEG C of preservations, extract RNA and purifying, get the above-mentioned viral RNA extracting solution of 1 μ L, add quantitative fluorescent PCR reaction system, utilize primer RBSDV-F:5 '-AGA GCT CTT CTA GTT ATT GCG-3 ' and RBSDV-R:5 '-TCA GCA AAA GGT AAA GGA ACG-3 ' according to fluorescent quantitative PCR program amplification RBSDV virus S7 fragment, increase complete, enter interpretation of result interface, take GAPDH as internal reference, compared with control group, obtain the RBSDV virus S7 of each sampling time point repetition of each kind and the relative expression quantity (RQ value) of reference gene,
5) RBSDV proliferation index Trendline is drawn: utilize each kind to be measured of multiple multiple RQ Data-Statistics and qualification scale to raise the RQ value average of the rear each sampling time point of poison, be ordinate zou with RQ value average, raise the rear cycle sampling time point of poison for X-coordinate, statistical study is carried out to data, obtains RBSDV proliferation index Trendline y=Ae
kx;
6) wheat breed to be measured is evaluated to the resistance level of wheat rosette stunt and resistance class according to RBSDV proliferation index Trendline formula: according to each kind RBSDV proliferation index Trendline formula y=Ae
kxevaluate the resistance level of wheat breed to be measured, namely the kind that the kind that in formula, k value is large is less than k value is low to RBSDV resistance, what kind k value to be measured was less than or equal to high resistance scale is high resistance kind, kind k value to be measured be greater than high resistance scale and in being less than or equal to anti-scale be in anti-kind, anti-scale during kind k value to be measured is greater than and be less than or equal to susceptible scale for susceptible variety, what kind k value to be measured was greater than susceptible scale is telegraphy kind.
2. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, is characterized in that: described step 1) and 6) in high resistance identify that scale is that indoor connect during poison is identified and disease-resistantly show that in local wheat variety resources, rank is 9%-11%; In anti-ly identify that scale rank is 23%-27%; Susceptiblely identify that scale rank is 47%-53%.
3. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, is characterized in that: described step 2) in the IAA strength of solution of suitable concentration be 1.5-2.5 μm of ol/L; Described hypoxia inducible method is that under seed is placed in liquid level, 5-10cm place cultivates.
4. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, is characterized in that: the obtaining step of the band poison small brown rice planthopper in described step 3) is:
3.1) RBSDV poison source obtains: gather the tender diseased plant of wheat children showing as wheat rosette stunt symptom from field, wheat rosette stunt grave illness district ,-70 DEG C of preservations;
3.2) virus mediator successive propagation is passed: land for growing field crops gathers and purifying small brown rice planthopper nymph at advanced age, raises and go down to posterity with wheat seedling;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out wheat rosette stunt diseased plant when raising poison, normal temperature places 3-5h makes diseased plant naturally launch, then closely wrap up with plastics bag with after water suction cotton parcel diseased plant root, guarantee that water can not flow out, then moved in crisper, the small brown rice planthopper that indoors artificial is raised is proceeded on the RBSDV poison source wheat in crisper, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred to wheat seedling raises and spends the phase of walking around to and be the malicious small brown rice planthopper of band.
5. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, is characterized in that: the ABA strength of solution of the suitable concentration in described step 3) controls as 40-60 μm of ol/L; The malicious small brown rice planthopper of quantitative inoculation band in described step 3) controls as a 6-10 worm/coleoptile.
6. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, is characterized in that: the periodic sampling time point in described step 4) is that RBSDV passes in malicious latter 4-10 days every a 1.5-2.0 days equally distributed 3-4 time point; The coleoptile of specific position is the position, middle of coleoptile length.
7. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, it is characterized in that: the method for the extraction RNA in described step 4) is: get appropriate blade and add liquid nitrogen and grind rapidly in mortar, add in TRI-Reagent to 1.5ml centrifuge tube in proportion, sample volume should more than TRI-Reagent volume 10%, 4 DEG C of 12000 × g low-temperature centrifugation 1.5min, then carefully shift supernatant in new centrifuge tube.
8. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, it is characterized in that: the method for the purifying RNA in described step 4) is: a) directly add a volume (95%-100%) alcohol to being dissolved in the supernatant liquor of TRI-Reagent, whirlpool mixes; B) transfer in adsorption column and filter (adsorption column is placed in collection tube), the then centrifugal 1min of 12000 × g, afterwards adsorption column is transferred in new collection tube; C) 400 μ l RNA Wash Buffer are added in centrifugal column, the centrifugal 1min of 12000 × g; D) the DNAase I cocktail buffer prepared by 80 μ l is directly added in centrifugal column, 25-37 DEG C of heating in water bath centrifugal column 15min, the centrifugal 30s of 12000 × g; E) add 400 μ l RNA Prewash in adsorption column, the centrifugal 1min of 12000 × g, collect the liquid filtered, repeat this step; F) add 700 μ l RNA Wash Buffer in adsorption column, the centrifugal 1min of 12000 × g, then from collection tube careful transfer adsorption column in a new RNase-free 1.5ml centrifuge tube; G) at least add 25 μ l DNase/RNase-Free Water in centrifuge tube matrix, maximum velocity centrifugation 1min, eluted RNA can directly utilize or be stored in-70 DEG C of Ultralow Temperature Freezers.
9. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 1, is characterized in that: contain in the every 20 μ L of the fluorescent quantitation reaction system in described step 4): the ddH of 2 × qPCR master mix (Promega) of 10 μ L, the cDNA template of 1.0 μ L, 8.2 μ L
210 μMs of primer RBSDV-R and RP of O and 0.8 μ L; Real-time fluorescence quantitative PCR amplification program is: 95 DEG C of denaturation 2min; 95 DEG C of sex change 15s, 60 DEG C of annealing 15s, 72 DEG C extend 20s, carry out 40 circulations altogether; 95 DEG C of sex change 15s, 60 DEG C of annealing 1min, 95 DEG C of sex change 15s.
10. the method by detecting coleoptile inner virus rate of propagation Fast Evaluation wheat rosette stunt varietal resistance according to claim 4, is characterized in that: described step 3.3) in RBSDV poison source wheat derive from step 3.1) in the tender diseased plant of wheat children showing as wheat rosette stunt symptom.
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