CN104830997A - Molecular identification method of rice black-streaked dwarf virus resistance - Google Patents
Molecular identification method of rice black-streaked dwarf virus resistance Download PDFInfo
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Abstract
The invention discloses a molecular identification method of rice black-streaked dwarf virus resistance. The molecular identification method comprises the following steps: (1) selecting identification scales; (2) performing hypoxia induction on to-be-measured rice and identifying coleoptiles of scale varieties; (3) inoculating viruliferous laodelphax striatellus to the coleoptiles, and then performing dark culture; (4) acquiring relative expression quantities (RQ values) of RBSDV virus S7 at cycle time points of various rice varieties and reference genes by using a fluorescent quantitative PCR process; (5) drawing an RBSDV proliferation index trend line; and (6) evaluating the resistance levels and resistance grades of to-be-measured rice varieties to rice black-streaked dwarf virus according to an RBSDV proliferation index trend line formula. By adopting the molecular identification method disclosed by the invention, the resistance levels and resistance grades of rice varieties to rice black-streaked dwarf virus can be accurately identified on a molecular level within a short time; and the molecular identification method is short in identification cycle and high in reliability, and has relatively large seedling raising application values.
Description
Technical field
The present invention relates to technical field of agriculture science, specifically a kind of qualification cycle be short, the molecular assay method of black streaked dwarf virus of rice resistance that reliability is high.
Background technology
Paddy rice is Angiospermae Monocotyledonae Cyperales Gramineae oryza plant.Paddy rice is one of topmost Three major grain crops, sown area accounts for 1/5 of grain acreage, and annual production about 4.8 hundred million tons, accounts for 1/4 of world food ultimate production, the population in the whole world more than 1/2nd take paddy rice as staple food, simultaneously one of topmost raise crop of Ye Shi China.Paddy rice originates in Tropical Asian, is one of world's staple food crop.China's rice growing face accounts for 1/4 of national food crop, and output then accounts for over half, and cultivation history has 6000-7000.Paddy rice is important food crop, except edible caryopsis, can starch processed, wine brewing, vinegar processed, rice bran can refine sugar, extracts oil, extract furfural, for industrial and pharmaceutical, rice straw is good feed and paper making raw material and braided material, and rice sprout and rice root can hyoscines, and paddy rice has critical role in Grain Development safety, processing and manufacturing in producing.
Black streaked dwarf virus of rice is a kind of virus disease caused by rice black-streaked dwarf virus (Rice black-streaked dwarf virus, RBSDV), is mainly that vector is propagated with small brown rice planthopper.Host has hybrid rice, conventional rice, barley, wheat, corn, millet, Chinese sorghum, amur foxtail, annual bluegrass, deceives grass, barnyard grass, Ma Tang etc. 28 kinds.In recent years, along with the change of cropping system and planting type, agricultural ecological are various, winter climate warms and the spread of susceptible variety, black streaked dwarf virus of rice in Jiangsu, Zhejiang, Hunan etc. economize extensive generation, becomes rapidly one of topmost rice disease in rice district, the middle and lower reach of Yangtze River.According to statistics, within 2007, black streaked dwarf virus of rice only occurs in partial area, Jiangsu Province, hazard area 30.8 ten thousand mu, rapid in the Huaibei to 2008, coastal and LX-river area spreads out, the whole province's onset area rises to 4,000,000 mu, wherein only the total crop failure field area of diseased plant rate more than 80% just reaches 30,000 mu, paddy 1.5 hundred million kilograms is about lost by average yield per mu 500 kilograms of preresearch estimatess, cause grain drop in production, peasant's reduction of income, huge loss is brought to agriculture production, the simultaneously also serious contusion kind grain enthusiasm of peasant, also the grain security in the whole nation is formed and threaten greatly.
At present, mainly use pesticide control to pass virus mediator small brown rice planthopper to the control of black streaked dwarf virus of rice, but due to amboceptor the quantity of insects' population large, cause prevention effect not good, and there is environmental pollution.And to cultivate disease-resistant variety be the most economic, the effective means of all kinds of disease of control.Screening, excavating and innovate Resistant gerplasm is 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 provide brand-new and tool.But black streaked dwarf virus of rice endangers serious due to it so far, can anti-uncurable disease, and the rice varieties major part that current production is promoted is not disease-resistant, is thus badly in need of cultivating and improveing out black streaked dwarf virus of rice resistant variety, fundamentally could reduces the loss that this disease is brought.Large-scale Screening Rice Germplasm Resources is the basis of cultivating resistant variety, and carrying out Resistance Identification to candidate's germ plasm resource is then important and necessary measure.
Current rice breeding screens disease-resistant variety, localization of disease resistance genes qualification resistance family all adopts field natural occurrence to identify, plant in land for growing field crops by each identification of species or strain community form, about each community tens strain, land for growing field crops conventional cultivation management, pass virus mediator small brown rice planthopper and naturally pass poison, treat that paddy rice manifests the cave sickness rate of each identification of species of illness " Invest, Then Investigate " (family) community, differentiate the difference of the anti-sense ability of rice varieties (family) as standard.But because the resistance of plant to natural enemy is divided into tolerance to insects, antibiosis and antixenosis three kind, various resistance classification tool is by different resistance mechanisms, and paddy rice is not also outside it.Different rice varieties (family) difference to small brown rice planthopper antixenosis makes the characteristics such as some physiology and morphology of different rice varieties (family), shows the difference taking food deflection to small brown rice planthopper.And the biography virus mediator of black streaked dwarf virus of rice is being just small brown rice planthopper, therefore, identified the disease resisting rice obtained by natural appraisal method, pest-resistant (antixenosis is strong) paddy rice may be actually and be not disease resisting rice.But if after carrying out the scale plantation of disease district with this pest-resistant not disease resisting rice improved variety, scale plantation causes passing virus mediator and is forced to select and make paddy rice disappear to the disease-resistant performance of vacation that small brown rice planthopper row becomes to producing, and may bring larger loss to agriculture production.
For getting rid of the impact of antixenosis enantiopathy qualification, quantitative paddy rice carries out artificial quantitatively inoculation band virus mediator and becomes a kind of inevitable.At present, removing that academy of agricultural sciences of Jiangsu Province Zhou Tong etc. utilizes artificial screening to identify malicious (RSV virus) small brown rice planthopper carries out artificial breeding, in room conditions to be quantitatively with malicious small brown rice planthopper to inoculate paddy rice certain hour to be measured specific breeding time in paddy rice, after cultivating paddy rice to be measured to the aobvious disease of morbidity, the resistance level of paddy rice to be measured to black streaked dwarf virus of rice is evaluated according to the severity that disease occurs, the method can overcome the interference of antixenosis in natural appraisal method, achieves the evaluation of science paddy disease-resistant accurately.But the Resistance Identification carrying out variety resources of rice needs a large amount of biography virus mediator small brown rice planthoppers, artificial screening nontoxic small brown rice planthopper artificial breeding scale amount is little, is difficult to the demand of the scale amount meeting varietal resistance qualification, limits 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 novel method 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 proves, to rice plant inoculation RBSDV, no matter to be in disease-resistant or susceptible rice plant body, RBSDV virus all detected, after rice plant infects RBSDV virus seedling stage in addition, symptom does not highlight, and can not determine its disease resistance, needs tillering regularity by the time could determine its disease resistance.Stem-winding, we study recently, and to find that paddy rice hypoxemia sprouts the RBSDV virus multiplication speed that the coleoptile produced inoculates after RBSDV virus in for some time coleoptile directly related with Resistance of Rice Varieties.Coleoptile is the taper shell-like thing outside paddy rice plumule, and be a sheath-like structure, coleoptile was the protective tissue of plant leaf originally, has the effect of more immature leaf and growing tip in protection plumule.Usual rice plant infects RBSDV virus and all passes poison by small brown rice planthopper seedling stage at rice seedling, and 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 Resistance of Rice Varieties, and our research finds to provide new approaches to the qualification of black streaked dwarf virus of rice disease resistance.
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 rice varieties is to the resistance level of black streak dwarf and resistance class.This authentication method, except can being applied to the excavation qualification of variety resources of rice, 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
To the object of the invention is for existing rice varieties, to the shortcoming such as black streaked dwarf virus of rice qualification cycle is long, qualification result is inaccurate, to provide the molecular assay method of the black streaked dwarf virus of rice resistance that a kind of qualification cycle is short, reliability is high.
The object of the invention is to solve by the following technical programs:
A molecular assay method for black streaked dwarf virus of rice resistance, 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 black streaked dwarf virus of rice Resistant expression, selecting three corresponding rice varieties as qualification scale;
2) coleoptile of hypoxia inducible paddy rice 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 each RBSDV virus S7 of rice varieties point cycle time and the relative expression quantity (RQ value) of reference gene: gather paddy rice to be measured according to some cycle time and identify the coleoptile 40-60g of scale specific position, 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) rice varieties to be measured is evaluated to the resistance level of black streaked dwarf virus of rice 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 rice varieties 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 variety resources of rice, rank is 7%-9%; In anti-ly identify that scale rank is 18%-22%; 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 tillering regularity paddy rice diseased plant showing as black streaked dwarf virus of rice symptom from field, black streaked dwarf virus of rice 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 rice seedling;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out black streaked dwarf virus of rice 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 length of time nymph that indoors artificial is raised is proceeded on the RBSDV poison source paddy rice in crisper, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred on young tender rice 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 described malicious small brown rice planthopper of quantitative inoculation band controls as a 5-8 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 small brown rice planthopper raise poison after the phase that walks around to be 23-27 days.
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 and accompanying drawing, the present invention is further illustrated.
A molecular assay method for black streaked dwarf virus of rice resistance, 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 black streaked dwarf virus of rice Resistant expression, select three corresponding rice varieties as qualification scale, wherein, high resistance identifies that scale is that indoor connect during poison is identified and disease-resistantly show that in local variety resources of rice, rank is 7%-9%, in anti-ly identify that scale rank is 18%-22%, susceptiblely identify that scale rank is 47%-53%;
2) coleoptile of hypoxia inducible paddy rice 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 tillering regularity paddy rice diseased plant showing as black streaked dwarf virus of rice symptom from field, black streaked dwarf virus of rice 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 rice seedling;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out black streaked dwarf virus of rice 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 length of time nymph that indoors artificial is raised is proceeded on the RBSDV poison source paddy rice in crisper, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred to and young tender rice seedling raises the phase that walks around to spending 23-27 days and be the malicious small brown rice planthopper of band;
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 5-8 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 RBSDV virus S7 of each rice varieties point cycle time and the relative expression quantity (RQ value) of reference gene: small brown rice planthopper passes maliciously terminates rear 4-10 days inherences and gather paddy rice 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) rice varieties to be measured is evaluated to the resistance level of black streaked dwarf virus of rice 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 rice varieties 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, utilize the authentication method of this invention to carry out black streak dwarf Resistance Identification to collecting the Some Rice Varieties resource obtained, concrete steps were as follows:
1) qualification scale is selected: connect disease-resistant performance in poison qualification according to indoor and black streaked dwarf virus of rice qualification scale is set, arrange that town rice 88, Huaihe River are glutinous 12 respectively, elegant water 04 3 rice varieties are as high resistance, middle resistant, susceptible qualification scale, wherein, town rice 88 connects in poison qualification in 2012-2014 indoor and disease-resistantly shows rank 12 in 152 Huaibei Dao Qu variety resources of rice colonies, glutinous 12 ranks 30 in Huaihe River, elegant water 04 rank 76;
2) coleoptile of hypoxia inducible paddy rice to be measured and qualification scale kind: each kind gets 18 seeds, 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 25 DEG C of lower 2.0 μm of ol/L of condition, 8cm 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: within 2014, gather the tillering regularity paddy rice diseased plant showing as black streaked dwarf virus of rice symptom ,-70 DEG C of preservations from field, black streaked dwarf virus of rice grave illness district, Jiangsu;
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 rice seedling Wu-Yu-Geng 3;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out black streaked dwarf virus of rice 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, indoors artificial is raised two age small brown rice planthopper nymph proceed on the RBSDV poison source paddy rice in crisper, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred to Wu-Yu-Geng 3 rice shoot raises and spends the phase of walking around in 25 days;
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 worms/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 50 μm of ol/L under being then positioned over 25 DEG C of conditions;
4) quantitative fluorescent PCR obtains each RBSDV virus S7 of rice varieties point cycle time and the relative expression quantity (RQ value) of reference gene: small brown rice planthopper passes poison and terminates gathered paddy rice to be measured in rear 4-10 days every 2 days and identify that the length position, middle of scale coleoptile is about 50g, the each time point 3 of each kind repeats,-70 DEG C of preservations, then RNA is extracted 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.) then quantitative fluorescent PCR, increasing complete, enter interpretation of result interface, is internal reference with GAPDH, compared with control group, obtains the relative expression quantity (RQ value) of RBSDV virus S7 that each sampling time point of each kind repeats and reference gene;
5) RBSDV proliferation index Trendline is drawn:
5.1) calculate paddy rice to be measured and identify that the RQ value average of malicious rear each sampling time point raised by scale: utilize each kind to be measured of the RQ Data-Statistics of 3 repetitions and qualification scale to raise the RQ value average of malicious rear each sampling time point, identify that scale RQ average statistics is as following table;
5.2) draw RBSDV proliferation index Trendline: with RQ value average for ordinate zou, raising the rear cycle time point of poison is X-coordinate, carries out statistical study to data, obtains RBSDV proliferation index Trendline y=Ae
kx, as accompanying drawing;
6) rice varieties to be measured is evaluated to the resistance level of black streaked dwarf virus of rice 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 rice varieties 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.
In this experiment, the k value of high resistance scale town rice 88 is calculated as 0.4141; In the k value in anti-scale Huaihe River glutinous 12 be calculated as 1.4188; The k value of susceptible scale show water 04 is calculated as 2.0132, as shown in drawings.The k value of kind to be measured compares to qualification scale and obtains corresponding resistance class, and we adopt authentication method of the present invention to identify a kind matter black streak dwarf resistance to following 12 rice varieties, and result is as table two;
By the qualification result of table two is fallen ill comparing of result with field multiple years black streak dwarf, 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 evaluate accurately kind of a matter black streak dwarf resistance coleoptile detection after seed germination, thus substantially reduce qualification time.
Can be found by embodiment, new Identification Method of the present invention, compared with authentication method in the past, just can be determined its resistance level by coleoptile, substantially reduce qualification cycle period after Seed Germination of Rice; Determine compared with the method for anti-perception with utilizing illness, after inoculation, from molecular level, coleoptile inner virus rate of propagation determines that rice germplasm is to the resistance level of black streaked dwarf virus of rice and resistance class by inquiry, 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. a molecular assay method for black streaked dwarf virus of rice resistance, 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 black streaked dwarf virus of rice Resistant expression, selecting three corresponding rice varieties as qualification scale;
2) coleoptile of hypoxia inducible paddy rice 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 each RBSDV virus S7 of rice varieties point cycle time and the relative expression quantity (RQ value) of reference gene: gather paddy rice to be measured according to some cycle time and identify the coleoptile 40-60g of scale specific position, 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) rice varieties to be measured is evaluated to the resistance level of black streaked dwarf virus of rice 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 rice varieties 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 molecular assay method of black streaked dwarf virus of rice 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 variety resources of rice, rank is 7%-9%; In anti-ly identify that scale rank is 18%-22%; Susceptiblely identify that scale rank is 47%-53%.
3. the molecular assay method of black streaked dwarf virus of rice 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 molecular assay method of black streaked dwarf virus of rice 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 tillering regularity paddy rice diseased plant showing as black streaked dwarf virus of rice symptom from field, black streaked dwarf virus of rice 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 rice seedling;
3.3) amboceptor raises poison: from-70 DEG C of refrigerators, take out black streaked dwarf virus of rice 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 length of time nymph that indoors artificial is raised is proceeded on the RBSDV poison source paddy rice in crisper, 25 DEG C raise malicious 48h after remove malicious source, small brown rice planthopper is transferred on young tender rice seedling raise spend the phase of walking around to be band malicious small brown rice planthopper.
5. the molecular assay method of black streaked dwarf virus of rice 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 5-8 worm/coleoptile.
6. the molecular assay method of black streaked dwarf virus of rice 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 molecular assay method of black streaked dwarf virus of rice 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 molecular assay method of black streaked dwarf virus of rice 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 molecular assay method of black streaked dwarf virus of rice 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 molecular assay method of black streaked dwarf virus of rice resistance according to claim 4, is characterized in that: described step 3.3) in small brown rice planthopper raise poison after the phase that walks around to be 23-27 days.
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